Medical organogel processes and compositions

ABSTRACT

Serial-solvent biomaterials are described. Embodiments include materials made in an organic solvent that are stripped of the solvent and used in a patient, where they imbibe water and form a hydrogel. These materials are useful for, among other things, delivering therapeutic agents, tissue augmentation, and radiological marking.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Ser. No. 61/566,768filed Dec. 5, 2011, which is hereby incorporated by reference herein.

TECHNICAL FIELD

The technical field generally relates to controlled release of drugs,and includes delivery of proteins from small particles.

BACKGROUND

Therapeutic agents require a means of delivery to be effective. Drugdelivery relates to administering a pharmaceutical compound to achieve atherapeutic effect in humans or animals. Delivery mechanisms thatprovide release of an agent over time are useful. Drug deliverytechnologies can help to modify a drug release profile, absorption,distribution or drug elimination for the benefit of improving productefficacy and safety, as well as patient convenience and compliance.

SUMMARY

Despite a great deal of research in these arts, the usefulness andsuccess of therapies using biologics, including proteins, continues tobe quite limited because of poor stability of the biologic in vivo.Despite conventional wisdom that proteins should not be exposed toorganic solvents in pharmaceutical processing techniques, it has beenobserved that many solvents can be used. Methods that use such solventsare described, including embodiments for two-solvent delivery systemswith the first solvent being an organic solvent in processing and thesecond solvent being physiological fluids in vivo.

An embodiment of the invention is a xerogel that comprises a proteinpowder, or other water soluble biologic powder, dispersed in a matrix ofthe xerogel. The xerogel may be hydrated at the point of use and placedin a tissue, where it controllably releases the protein over time. Thisembodiment and others are detailed below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts formation of a biomaterial;

FIG. 1B depicts a microstructure of the biomaterial of FIG. 1A;

FIG. 1C depicts a microstructure of an alternative embodiment of abiomaterial;

FIG. 2A is a plot of HPLC data showing release of ovalbumin over time inphysiological solution at 37° C.;

FIG. 2B is a plot of the data of FIG. 2A after being normalized to theprotein level at complete dissolution of the hydrogel;

FIG. 3 is a plot of HPLC data showing release of ovalbumin over time inphysiological solution at pH 8.5 and 37° C. and in physiologicalsolution at pH 7.4 and 37° C. Data are normalized to the protein levelat complete dissolution;

FIG. 4 is a plot of HPLC data showing release of IgG over time inphysiological solution at 37° C.;

FIG. 5 is a plot of the data of FIG. 4 after being normalized to theprotein level at complete dissolution of the hydrogel;

FIG. 6 is a plot depicting a calculated release profile of albumin froma combination of hydrogel vehicles;

FIG. 7 is a plot depicting a calculated release profile of albumin froma combination of hydrogel vehicles;

FIG. 8 is an illustration of various sites at or near an eye forapplication of a biomaterial;

FIG. 9A is an illustration of a method for placing a biomaterial in aneye, and depicts a process of inserting a needle into an eye; and

FIG. 9B depicts various examples of sites to receive the biomaterial inthe eye of FIG. 9A.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

An embodiment of the invention is a xerogel that comprises a proteinpowder, or other water soluble biologic powder, dispersed in a matrix ofthe xerogel. The xerogel may be hydrated at the point of use and placedin a tissue, where it controllably releases the protein over time. Thepowder contains fine particles of protein. The xerogel matrix, uponhydration, is a hydrogel made of a crosslinked matrix. The protein is ina solid phase and is substantially not soluble until the matrix beginsto erode, thereby allowing the protein to go into solution. The matrixprotects the protein from cells, enzymatic denaturation, and unwantedlocal reactions. The protein is in a substantially solid phase untilreleased by gradual solvation and is thus protected from denaturation,autohydrolysis, proteolysis, and local chemical reactions that can causea loss of effectiveness or create antigenicity.

FIG. 1A depicts an embodiment of this process, which is started withprotein particles 100 that have been prepared by conventional means topreserve protein secondary and, if present, tertiary or quaternarystructure. These are combined with precursors 102, 104, into organicsolvent 106. The mixture is processed to achieve the desired shape ofthe biomaterial, e.g., by casting 108, as rod 110, as particles and/orspheres 112, and molded shapes 114. The solvent is stripped out of theshapes and the materials will form hydrogels when exposed to water. Theentire process, until the point where the xerogel is actually used witha patient, may be performed in an absence of water and/or in an absenceof hydrophobic materials. FIG. 1B depicts a microstructure of abiomaterial 120 made by this process. The structure is representative ofthe material across the process of its manufacture and use: organogel,xerogel, and then hydrogel. The crosslinked matrix is made of precursors124 that have been covalently reacted with each other. Particles 124 ofa water soluble biologic are dispersed within the matrix. The matrix isa continuous phase and the particles are spread out inside it and arethe discontinuous phase, also referred to as the dispersed phase.

Alternative embodiments involve using block copolymer precursors thatare physically crosslinked by formation of hydrophobic domains, asdepicted in FIG. 1C. The biomaterial 130 has biologic particles 132dispersed in the matrix. The precursors have hydrophilic blocks 134 andhydrophobic blocks 136. The hydrophobic blocks 136 self assemble to formhydrophobic domains 138, which create physical crosslinks between theprecursors. The term physical crosslink means a non-covalently bondedcrosslink. Hydrophobic domains are one such example, as well as thehard-and-soft segments of a polyurethane or other segmented copolymers.Ionic crosslinks are another example. The term crosslink is wellunderstood by artisans, who will immediately be able to distinguishcovalent crosslinks from physical cross links, as well as the subtypesof physical crosslinks such as ionic, hydrophobic, and crystallinedomains.

Other drug delivery approaches have encapsulated proteins with, forexample, liposomes or micelles, or made nanoparticles that use polymersor other agents in creation of the particles. Protein delivery in ahydrogel has been generally directed to sequestering the proteins fromthe hydrogel: for example, by placing the hydrogel in a liposome,micelle, or in a mixture with a binding agent such as a polymer. Otherapproaches have been directed to directly adsorbing materials toproteins so as to inhibit their dissolution. Another approach was toprecipitate proteins in the delivery process, as disclosed in U.S.Publication No. 2008/0187568. Other approaches use hydrogels withsoluble proteins dispersed through the hydrogel, with hydrogel erosioncontrolling the release.

Despite all of these efforts, the usefulness and success of sustainedrelease therapies using biologics, including proteins, is limitedbecause the stability of the biologic in vivo tends to be poor. And aloss of conformation can lead not only to a loss of efficacy, but it canbe detrimental by causing unwanted effects or eliciting an immuneresponse. Despite very many efforts, there have been no generallyapplicable solutions effective enough to have real-world clinical value,as documented in Wu and Jin, AAPS Pham Sci Tech 9(4): 1218-1229 (2008).

Surprisingly, however, the embodiments provided herein show that proteinor other biologic solubility and release from a matrix can be controlledby disposing a biologic as a solid-phase particulate in a suitablematrix so that these other approaches involving polymers, encapsulants,binders, and the like are not needed. Further, the biologic resistsdenaturation even in aqueous in vivo environments. The particulates inthe matrices are water soluble but, despite not having any coatings orthe like, are slow to dissolve and their dissolution in physiologicalsolution, which would normally be measured in minutes or hours, can beextended to days, weeks, or months. Moreover, another unexpected andsurprising result has been observed: namely, that the biologics do nottend to aggregate even though they are necessarily present at very highconcentrations within the matrix. It seems that the biologics come offof the particles very slowly. A first theory of operation, to which theinvention is not to be limited, is that the molecular strands of thematrix which are made of highly mobile polymers—for example polymerssuch as polyethylene glycol (PEG) or polyethyleneimine—form an exclusionvolume around themselves, which limits the solubility of any othermacromolecule in the immediate vicinity. This structural attribute notonly confines the proteins in the solid phase by physical entrapmentwithin the matrix, but also limits the dissolution of the macromolecule,so that the protein particle is unable to move into solution; as theparticles and proteins begin to swell by solvation with water, they arerestrained by the matrix until the matrix is at least partiallydissolved. Thus, as the crosslink density decreases and the molecularstrands move further apart, gradual dissolution of the entrappedmacromolecule particle is facilitated. These processes thus provide anunexpected and surprising result: the biologics stay in the solid phaseuntil they are getting relatively close to the time of their releasefrom the matrix: consequently, the protein or other biologic is stablebecause it is not exposed to the detrimental effects of being insolution for a long time. Release is also restricted by the diffusion ofthe macromolecule out from the matrix and is influenced by the molecularweight of the macromolecule as well as the characteristics of the matrixforming polymers. A second theory of operation is complementary to thefirst and is likewise not a mechanism to which the invention is to belimited: the molecular strands of the matrix are associating with watermolecules near the proteins such that the proteins are unable todissolve. This second theory is applicable to polymers with highlymobile, hydrophilic, linear chains such as PEG. Besides PEG, other watersoluble polymers or copolymers that exhibit an exclusion volume effectwith the selected protein can be chosen. For instance, polymers such aspolyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone (PVP), andpolyhydroxyethylmethacrylate (PHEMA) will generally have such an effect.Some polysaccharides also have these effects. PEG and/or these otherpolymers can also be incorporated as solids in the organogel. They willsolubilize in the presence of water, i.e., in the hydrogel. Moreover,non-crosslinked PEG and/or PEG copolymers such as a PLURONIC beadditives that can be trapped in the hydrogel along with the protein toenhance the exclusion volume effect, thereby keeping the proteins in thesolid state.

An aspect of the systems disclosed herein relates to a large increase incontrol over the time of release caused by placing protein particles ina hydratable xerogel. Examples 1-2 detail processes used to formxerogels containing particles of water soluble biologics. The proteinsalbumin and immunoglobulin (IgG) were used to model a water solubletherapeutic agent protein. Powders of these proteins were prepared. Thepowder particles were combined with hydrogel precursors in organicsolvents to form an organogel. Tables 1-5 of Example 1 set forthexamples of the organogels comprising the dispersed protein powder. Theorganogels were broken up and sieved into collections of particles thatwere evacuated of organic solvents to form xerogels. Working Example 2documents release of the proteins from the xerogels.

As illustrated in FIGS. 2-5, the proteins were fully released;unexpectedly, there was no detectable reaction of the organogelprecursors with the proteins that prevented them from being solubilizedas the matrix degraded. In fact, these proteins, and proteins ingeneral, contained amine and thiol functional groups that arepotentially very reactive towards strong electrophiles such as theelectrophilic precursors that were used. Although a reaction with theseelectrophilic functional groups was expected, the lack of reactionindicates that these reactions were prevented by leaving the proteins ina non-dissolved or substantially solid phase while the gel-formingprecursors were in a liquid phase prior to gelation. The release curvesshowed good control over the rate of release, and ranged from a quickrelease of hours to months of release.

Moreover, the rate and kinetics of release may be further controlled bycombining the various sets of particles with each other, as illustratedin FIGS. 6 and 7. These demonstrate a substantially zero order release,which is the ability to deliver a drug at a rate which is independent oftime and the concentration of drug within a pharmaceutical dosage formis desirable. A zero order release mechanism ensures that a steadyamount of drug is released over time, minimizing potential peak/troughfluctuations and side effects, while maximizing the amount of time thedrug concentrations remain within the therapeutic window (efficacy).

Processes and Materials for Preparing a Organogel-Hydrogel, Two-SolventDelivery System for Water Soluble Biologics

A first embodiment involves forming covalently crosslinked matrices. Afine powder of a water soluble biologic is prepared and suspended in anorganic solvent that does not solvate the water soluble biologic, e.g.,protein. The term powder is used broadly herein to refer to a collectionof dry particles. The term particles is broad and includes spheres,teardrop-shapes, small rods and other irregular shapes. In general, thepowder has been processed to provide a controlled particle compositionwith a known size, shape, and distribution (variance from a mean oraverage) thereof. Protein powders typically contain stabilizing sugarssuch as sucrose or trehalose. These sugars are generally water solubleand not organic soluble. It was found that these will remain with theprotein through the process until the point of hydration to form thehydrogel. Matrix precursors are prepared that have the capacity to forma crosslinked organogel by reacting with each other in the organicsolvent. The precursors are chosen to be soluble in the organic solvent.The precursors and water soluble biologic powder are mixed in theorganic solvent so that the water soluble biologic particles aredispersed through the matrix that forms upon formation of covalent bondsbetween the precursors. The matrix formed in the organic solvent isreferred to as an organogel. The solvent is removed to form a xerogel.Upon hydration in water the matrix forms an internally covalentlycrosslinked hydrogel. This process is a serial two-solvent processbecause the organic solvents have to be effective with the biologic andthe precursor(s), strippable (i.e., removable without leavingpharmaceutically unacceptable residue), but the precursors have to beeffective in an in vivo aqueous environment. At no time is the proteinexposed to both organic and aqueous phases. Exposure of proteins inaqueous solution to interfaces such as with organic liquids or solids orair bubbles is believed to contribute to protein adsorption anddenaturation. The organogel to xerogel to hydrogel serial processeliminates the possibility of interface exposure, i.e., embodimentsinclude processes as described herein being performed without exposureof a water soluble biologic to an interface between any combination ofthe following: air, gas, water, organic solvent.

Another embodiment is the formation of a covalently crosslinked gel(also referred to herein as a pseudo-organogel) by employing liquidreactive polymers as matrix precursors. Matrix precursors are preparedthat have the capacity to form a crosslinked organogel by reacting witheach other in the absence of organic solvent, e.g. when in the moltenstate. The precursors and water soluble biologic powder are mixed at atemperature high enough to liquefy the precursor polymers, but lowenough to maintain protein stability. Examples of such temperatures arefrom about 10° C. to about 75° C., or up to about 60° C. or up to about75° C.; artisans will immediately appreciate that all values and rangesbetween the explicitly stated values are contemplated and areincorporated herein as if written in detail. Mixing conditions areemployed such that the water soluble biologic particles are dispersedthrough the matrix that forms upon formation of covalent bonds betweenthe precursors. The reaction is thus performed in a melt of thepolymers, with the term melt meaning that no solvents are present. Theremay be, however, other materials in the melt, e.g., biologics, sugars,proteins, buffers. Embodiments include material and a process of makinga medical material comprising forming a gel around a powder of a watersoluble biologic, with the powder being dispersed in the gel, whereinforming the gel comprises preparing a melt of one or more precursors andcovalently crosslinking the precursors. Said gel may have a largeportion of its volume occupied by the biologic or other solids (e.g.,sugars, buffer salts), e.g., from about 30% to about 95% v/v; artisanswill immediately appreciate that all values and ranges between theexplicitly stated values are contemplated and are incorporated herein asif written in detail, e.g., at least 30% v/v or from about 40% to about75%.

Another embodiment of the serial two-solvent process involves forming acrosslinked material having physical crosslinks. One such embodimentuses block copolymers as precursors. The precursors have a lyophilic(solvent-loving) block and a lyophobic (solvent-hating) block. Theseprecursor(s) are added to the organic solvent and from a physicallycrosslinked matrix. The blocks (also referred to as segments) thatprecipitate in a specific organic solvent to form the organogel may ormay not be the same segments that precipitate in water to form thehydrogel. After stripping the solvent, the resultant xerogel forms ahydrogel in aqueous solution because one or more block or segmentportions are hydrophobic and one or more block or segment portions arehydrophilic. A related embodiment uses two organic solvents: the blockcopolymeric precursor is dissolved in a first organic solvent. Thecopolymer solution is then mixed with a second organic solvent that ismiscible with the first solvent, but is a non-solvent for the at leastone of the segments of the copolymer. The lyophobic domains form theorganogel. Another embodiment uses the first and second organic solventand uses the second organic solvent to also precipitate the biologic,such that the organogel and the particles of the biologic are formed atthe same step.

Another embodiment of the serial two-solvent process involves thermalgelation. A precursor that transitions from solution to an organogel inthe organic solvent at temperatures in a range of about −20° C. to about70° C. is placed with the biologics in the organic solvent at atemperature wherein the precursor is in solution. The solution is thencooled to a second temperature below the gelation point, and theprecursor forms an organogel. Accordingly, a process for making anorganogel is to heat a solvent to dissolve a copolymer and then cool thesolution to precipitate at least one of the segments of the copolymer.The solvent is then stripped to make a xerogel. The precursors arechosen so that the xerogel is a hydrogel at physiological temperatures.

Block copolymers adaptable for use in these processes include manycopolymers of PEG. The PEG is hydrophilic and is lyophilic for manyorganic solvents. Other hydrophilic polymers and polymeric segments arepolyvinyl alcohol, polyacrylic acid, polymaleic anhydride, PVP, PHEMA,polysaccharides, polyethylene imine, polyvinyl amine polyacrylamide(s),and the like. The other block is chosen to be hydrophobic and lyophobicfor the organic solvent. Examples of these other blocks are:polybutylene terephthalate (PBT), polylactic acid, polyglycolic acid,polytrimethylene carbonate, polydioxanone, and polyalkyl ethers such aspolypropylene oxide (PLURONICS, POLOXAMERS). The copolymers may have oneor more of each kind of block.

These processes may be performed so that the water soluble biologicnever contacts water from the time it is initially prepared until placedin vivo. The water soluble biologic may be further processed so that,once obtained in a purified form at the source or a manufacturing site,it is not thereafter dissolved in and/or is never exposed to waterduring the gel manufacturing process. Exposure to water can cause avariety of problems. One problem is that a protein will undergohydrolysis over time so that it is slowly degraded. Another problem isthat a protein, once it is in solution, can rearrange or formquasi-stable aggregates such as dimers or trimers.

Embodiments of the inventions include these process performed in theabsence of hydrophobic polymers and/or hydrophobic solvents. Theembodiments that require a hydrophobic block polymer can not beperformed in a hydrophobic-free process, but the artisan can readilydiscern which processes are applicable. One embodiment provides forhydrophilic precursors to be covalently crosslinked in the organicsolvent in the presence of a biologic particle and an absence ofhydrophobic materials, both at the organogel steps and subsequent steps.In some embodiments a solvent that is hydrophobic might be presentwithout detriment, depending on the solvent, so embodiments include anabsence of hydrophobic materials other than solvents; and/or an absenceof hydrophobic polymers; and/or an absence of hydrophobic polymersegments.

Conventional wisdom teaches that organic solvents generally denatureproteins. Some life sciences processes can tolerate some degree ofdenaturation, for example, in diagnostic or analytical settings. In themedicinal arts, however, even a small degree of denaturation isundesirable. Denatured proteins can exhibit a wide range ofcharacteristics, from loss of solubility to communal aggregation.Communal aggregation involves aggregation of the hydrophobic proteins tocome closer to each other to reduce the total area exposed to water. Areduction in distance can cause permanent or quasi-stable associations.When a protein is denatured, its secondary and tertiary structures arealtered but the peptide bonds of the primary structure between the aminoacids are generally left intact.

Surprisingly, however, it has been discovered that proteins left in asolid phase can be exposed to certain organic solvents without extensivedenaturation. Fully anhydrous organic solvents handled under anhydrousconditions are preferred. Denaturation from exposure to organic solventsmay happen when the protein is already in an aqueous solution and/or ifthe organic solvent, or organic/aqueous mixed solvent (e.g.ethanol/water), has a propensity to dissolve or even in a limited way,swell, the protein particle. Protein-solvent compatibility can beestablished experimentally by exposure followed by characterizationtesting to determine if the protein has been denatured and/or undergonesubstitution or alteration of one or more chemical groups. Organicsolvent compatibility can be tested simply by immersing the subjectprotein in the subject solvent for an appropriate period of time,removing the protein, such as by filtration and vacuum drying, and thentesting for recovery of the protein by HPLC or other appropriateanalytical method. Solvents most likely to leave the protein unharmedare anhydrous and hydrophobic, but must also be good solvents for thegel forming precursor molecules. In the case of polyethylene glycol(PEG) precursors, solvents such as methylene chloride and dimethylcarbonate have been employed. Other solvents such as acetone (oracetone/water), ethyl acetate, tetrahydrofuran, may also be useful.Supercritical fluid solvents such as carbon dioxide may also be usefulfor forming organogels.

The precursors are described in detail elsewhere herein. Many usefulprecursors are available as a plurality of precursors. A first precursoris added to the solvent-protein mixture, followed by a second precursorthat is reactive with the first precursor to form crosslinks. The firstprecursor may be chosen to have only those functional groups that areunreactive to form covalent bonds with a protein in the absence offurther chemical components. Proteins have amines and thiols that may beused to react with certain electrophilic functional groups to formcovalent bonds, as well as carboxyls and hydroxyls that are availablefor other chemical reactions. The precursor may accordingly be chosen tobe unreactive with these functional groups. For example, the precursormay have amines and/or thiols and/or hydroxyls and/or carboxyls and beunreactive with proteins. Accordingly, an embodiment of the inventioninvolves adding a first protein-unreactive precursor to aprotein-organic solvent mixture and then adding a second precursor thatis reactive with the first precursor.

The water soluble biologic particles may be free of one or more of:binders, fatty acids, hydrophobic materials, surfactants, fats,phospholipids, oils, waxes, micelles, liposomes, and nanocapsules. Theorganogel or xerogel comprising the water soluble biologic particles mayalso be free of one or more of the same. The protein or other watersoluble biologic in the xerogel may all be in a solid phase, may be allcrystalline, partially crystalline, or essentially free of crystals(meaning more than 90% free of crystals w/w; artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated).

The xerogel-water soluble biologic material may be formed in a desiredshape. One method is to react the precursors in a mold that has thedesired shape. The shape is removed from the mold before or afterremoval of the solvent. The material may also be fragmented intoparticles, as described in more detail elsewhere herein.

After formation of the matrix in the organic solvent, the solvent may beremoved to form the xerogel. Potential processes include, e.g.,precipitation with non-solvent, nitrogen sweep drying, vacuum drying,freeze-drying, a combination of heat and vacuum, and lyophilization.

If molten precursors are used in the absence of a tertiary solvent,there is no need to employ any solvent removal process. Upon cooling thematerial forms a rubbery solid (if above Tm), a semirigidsemicrystalline material (if below Tm) or a rigid glassy solid (if belowTg). These materials are more dense than xerogels formed from organicsolvents. When filled with particles of other materials, e.g.,therapeutic agents, buffer salts, visualization agents, they can behighly porous, since the solid particles create and fill the pores.

All of these processes may be performed without the water solublebiologic. Materials, including particles, have usefulness for manyapplications without the biologic. Uses include, e.g., tissueaugmentation, fillers, and tissue separations in radiotherapy.

Moreover, all of these processes may be performed with additional agentsinstead or, or additionally with, the biologics. Such additional agentsinclude visualization agents visible to a naked eye and radiopaqueagents or materials.

Particles Preparation

The organogel may be formed and then reduced to particles that aresubsequently treated to remove the organic solvent or solvents to form axerogel. For an injectable form, the organogel can be macerated,homogenized, extruded, screened, chopped, diced, or otherwise reduced toa particulate form. Alternatively, the organogel can be formed as adroplet or a molded article containing the suspended protein particles.

One process for making organogel particles involves creation of a matrixthat is broken up to make organogel particles. Thus matrices are madewith precursors as described herein and are then broken up. Onetechnique involves preparing the organogel with protein particles andgrinding it, e.g., in a ball mill or with a mortar and pestle. Thematrix may be chopped or diced with knives or wires. Or the matrix maybe cut-up in a blender or homogenizer. Another process involves forcingthe organogel through a mesh, collecting the fragments, and passing themthrough the same mesh or another mesh until a desired size is reached.

The water soluble biologics, e.g., proteins are prepared as particlesbefore dispersal into the organogels. Multiple protein particulationtechnologies, such as spray drying or precipitation exist and may beemployed provided the protein of interest is compatible with suchprocessing. An embodiment of particle preparation involves receiving thebiologic without substantial denaturation, e.g., from a supplier oranimal or recombinant source. The solid phase is a stable form for theprotein. The protein is lyophilized or concentrated or used as received.The protein is then prepared as a fine powder without denaturation byprocessing it in a solid state and avoiding high temperatures, moisture,and optionally in an oxygen free environment. Powders may be preparedby, for example, grinding, ball milling, cryomilling, microfluidizing ormortar-and-pestle followed by sieving a solid protein. The protein mayalso be processed in a compatible anhydrous organic solvent in which theprotein in question is not soluble, while keeping the protein in a solidform. Particle size reduction to the desired range may be achieved by,for example, grinding, ball milling, jet milling of a solid proteinsuspension in a compatible organic solvent. High shear rate processing,high pressure, and sudden temperature changes should be minimized asthey lead to protein instability. Accordingly, care must be taken tohandle the protein or other water soluble biologic in a manner thatavoids damage, and the use of routine processes for making particlesshould not be assumed to be suitable and are not to be expected to beuseful without suitable re-engineering and testing of the results.

The term powder of the protein refers to a powder made from one or moreproteins. Similarly, powders of water soluble biologics are powdershaving particles made of one or more water soluble biologics. Theproteins in a protein particle or the biologics in a biologics particleare associated with each other to provide mechanical integrity andstructure to the dry particle even in the absence of binders orencapsulants. These powders are distinct from protein or biologicdelivery using an encapsulation or approach such as a liposome, micelle,or nanocapsule other technique that substantially encapsulates a proteinor biologic. The powders and/or xerogels or hydrogels that contain themmay be free of encapsulating materials and be free of one or more of aliposome, micelle, or nanocapsule. Further, a protein particle or awater soluble biologic particle may be made that is free of one or moreof: binders, non-peptidic polymers, surfactants, oils, fats, waxes,hydrophobic polymers, polymers comprising alkyl chains longer than 4 CH₂groups, phospholipids, micelle-forming polymers, micelle-formingcompositions, amphiphiles, polysaccharides, polysaccharides of three ormore sugars, fatty acids, and lipids. Lyophilized, spray dried orotherwise processed proteins are often formulated with sugars such astrehalose to stabilize the protein through the lyophilization or otherprocesses used to prepare the proteins. These sugars may be allowed topersist in the particle throughout the organogel/xerogel process. Theparticles may be made to comprise between about 20% and about 100% (dryw/w) protein; artisans will immediately appreciate that all the rangesand values within the explicitly stated ranges are contemplated, e.g.,about 50% to about 80% or at least 90% or at least about 99%.

The particles of biologics or the particles or organogels or theparticles of the xerogels may be separated into collections with adesired size range and distribution of sizes by a variety of methods.Very fine control of sizing is available, with sizes ranging from 1micron to several mm, and with a mean and range of particles sizes beingcontrollable with a narrow distribution. Artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated, e.g., from about 1 to about 10 μm or from about1 to about 30 μm. About 1 to about 500 microns is another such rangethat is useful, with sizes falling throughout the range and having amean sizing at one value within the range, and a standard deviationcentered around the mean value, e.g., from about 1% to about 100%. Asimple method for sizing particles involves using custom-made orstandardized sieve mesh sizes. In addition to standard U.S. and Tylermesh sizes, sieves are also commonly used in the Market Grade, MillGrade, and Tensile Bolting Cloth. Materials forced through meshes mayshow deformation so that the particle size is not precisely matched tomesh sizes; nonetheless, mesh sizes may be chosen to achieve a desired aparticle size range. Particle size analyzers where the protein particleis dispersed in an organic or oil phase are commonly used. Microscopy isalso commonly used to determine particle size. A spheroidal particlerefers to a particle wherein the longest central axis (a straight linepassing through the particle's geometric center) is no more than abouttwice the length of other central axes, with the particle being aliterally spherical or having an irregular shape. A rod-shaped particlerefers to a particle with a longitudinal central axis more than abouttwice the length of the shortest central axis. Embodiments includemaking a plurality of collections of particles, with the collectionshaving different rates of degradation in vivo, and mixing collections tomake a biomaterial having a degradation performance as desired.

Delivery of Water Soluble Biologics without Denaturation

These processes may be performed with a protein or other water solublebiologics. These include peptides and proteins. The term protein, asused herein, refers to peptides of at least about 5000 Daltons. The termpeptide, as used herein, refers to peptides of any size. The termoligopeptide refers to peptides having a mass of up to about 5000Daltons. Peptides include therapeutic proteins and peptides, antibodies,antibody fragments, short chain variable fragments (scFv), growthfactors, angiogenic factors, and insulin. Other water soluble biologicsare carbohydrates, polysaccharides, nucleic acids, antisense nucleicacids, RNA, DNA, small interfering RNA (siRNA), and aptamers.Descriptions herein are often set forth in terms of proteins but themethods are generally applicable to other water soluble biologics.

Proteins are easily denatured. As described herein, however, proteinsmay be delivered substantially without denaturation, including the casewherein no binders, lipophilic materials, surfactants, or otherprophylactic components are used. The term substantially withoutdenaturation refers to a protein processed into a particle withoutmodification of the protein's chemical structure (without addition ofchemical groups or changes of the existing chemical groups) and withoutchanges to the protein's conformation, i.e., secondary and/or tertiaryand/or quaternary structure. The term substantially, in this context,means that no significant differences (p-value<0.05) between processedproteins and control proteins are observed for an averaged test group astested under routine conditions by enzyme-linked immunosorbent assay(ELISA) for epitope denaturation and by isoelectric focusing (IEF) for ashift of more than 0.2 in isoelectric point (pI), see U.S. Ser. No.13/234,428, which is hereby incorporated by reference herein for testingor protein stability and all purposes; in case of conflict, the instantspecification controls. A primary protein structure refers to the aminoacid sequence. To be able to perform their biological function, proteinsfold into one or more specific spatial conformations, driven by a numberof non-covalent interactions such as hydrogen bonding, ionicinteractions, Van Der Waals forces, and hydrophobic packing. The termsecondary structure refers to the local protein structure, such as localfolding. The tertiary structure refers to a particular three-dimensionalconformation, including folding. A protein that has secondary and/ortertiary structure thus exhibits local and general structuralorganization. In contrast, a linear peptide that has no particularconformation does not have secondary and/or tertiary structure. The termnative means as found in nature in vivo, so that proteins may beprocessed into particles and released in a native conformation.

Proteins may be tested for denaturation by a variety of techniques,including enzyme-linked immunosorbent assay (ELISA), isoelectricfocusing (IEF), size exclusion chromatography (SEC), high-pressureliquid chromatography (HPLC), circular dichroism (CD), and FourierTransform Infrared Spectroscopy (FTIR). These tests report parameterssuch as changes in molecular weight, change in end groups, changes inbonds, changes in hydrophobicity or volume exclusion, andrevelation/hiding of antigenic sites. In general, a test by IEF andELISA may be designed that is adequate to show native conformation afterprocessing, although other tests and test combinations may alternativelybe used.

Experimentation has shown that a number of factors can be controlledthat contribute to processing and delivery of a protein withoutdenaturation. The protein may be prepared as a powder, with the powderparticle size being chosen in light of the size of the ultimateorganogel. All organic solvents for the proteins may be chosen so thatthe proteins are not solvated by the organic solvents and are compatiblewith the protein. Another factor is oxygen, and elimination of oxygen ishelpful in processing to avoid denaturation. Another factor is chemicalreactions. These may be avoided by keeping the protein in a solid phaseand free of solvents that dissolve the protein until such time as theprotein is implanted.

One embodiment of particle preparation involves receiving a proteinwithout substantial denaturation, e.g., from a supplier or animal orrecombinant source. The protein is lyophilized, spray dried orconcentrated or used as received. The protein is then prepared as a finepowder without denaturation by processing it in a solid state andavoiding high temperatures, moisture, and optionally in an oxygen freeenvironment. Powders may be prepared by, for example, grinding, ballmilling, or mortar-and-pestle a solid protein.

Making a protein agent or other water soluble biologic agent into aparticle can be a useful first step for delivery of the agent from asolid phase. It is not, however, a sufficient step for achieving awell-controlled release from a matrix, or effective release over anextended period of time. Upon implantation, however, the particle willtend to be quickly dissolved as water contacts the particle and solvatesthe agents. In the case of a particle in a hydrogel, for instance, waterpermeates the hydrogel and contacts the particles. Unexpectedly,however, it is possible to prevent the water soluble biologic agents inthe particles in the hydrogel from dissolving. Some mechanisms for doingso are set forth herein but are not to be used to limit the inventionsto particular theories of action. One mechanism is apparently related tousing a matrix that prevents the agents from moving away from theparticle. And, even if a molecule of the agent dissolves, it is kept atthe local site and will saturate the local site to prevent furthersolvation of other agent molecules. Another mechanism relates to thesolvation of the matrix, which competes for water with the agents thatare potentially soluble, with the matrix having a volume exclusioneffect for interfering with agent solvation.

These mechanisms relate to achieving a spacing between molecular strandsof the matrix that is dense. The crosslinking density of the organogelmatrix (and thus the xerogel and the hydrogel matrix) is controlled bythe overall molecular weight of the precursor(s) used as crosslinker(s)and other precursor(s) and the number of functional groups available perprecursor molecule. A lower molecular weight between crosslinks such as500 will give much higher crosslinking density as compared to a highermolecular weight between crosslinks such as 10,000. The crosslinkingdensity also may be controlled by the overall percent solids of thecrosslinker and functional polymer solutions. Yet another method tocontrol crosslink density is by adjusting the stoichiometry ofnucleophilic functional groups to electrophilic functional groups. A oneto one ratio leads to the highest crosslink density. Precursors withlonger distances between crosslinkable sites form gels that aregenerally softer, more compliant, and more elastic. Thus an increasedlength of a water-soluble segment, such as a polyethylene glycol, tendsto enhance elasticity to produce desirable physical properties. Thuscertain embodiments are directed to precursors with water solublesegments having molecular weights in the range of 2,000 to 100,000;artisans will immediately appreciate that all the ranges and valueswithin the explicitly stated ranges are contemplated, e.g. 10,000 to35,000. The solids content of the hydrogel can affect its mechanicalproperties and biocompatibility and reflects a balance between competingrequirements. A relatively low solids content is useful, e.g., betweenabout 2.5% to about 20%, including all ranges and values there between,e.g., about 2.5% to about 10%, about 5% to about 15%, or less than about15%. Artisans will appreciate that the same materials may be used tomake matrices with a great range of structures that will have highlydistinct mechanical properties and performance, such that theachievement of a particular property should not be merely assumed basedon the general types of precursors that are involved.

Delivery of Water Soluble Biologics and Other Therapeutic Agents

Various water soluble biologics and/or other therapeutic agents may bedelivered with the systems described herein. The xerogel particlescontaining protein powders may be used to deliver a water solublebiologic and/or other therapeutic agent. The particles may beadministered inside a xerogel. The xerogel may be a preformed structure,e.g., having at least 2 cm³ of volume (artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated, e.g., about 2 to about 20 cm³) or be acollection of particles. Alternatively, the xerogel particles may beadministered directly, or in a pharmaceutically acceptable binder orcarrier. Other materials may comprise the xerogel particles. Watersoluble agents are one category of agents that may be delivered aspowders within the xerogel. Other drugs may also be mixed into thexerogels, or with the xerogels, such as hydrophobic agents or smallmolecule drugs (water soluble or hydrophobic).

Proteins are a category of water soluble agents. The xerogel particlesmay be processed so that the proteins are incorporated and releasedwithout substantial denaturation and/or in their native conformation.Some anti-vascular endothelial growth factor (anti-VEGF) agents aretherapeutic agent proteins. Anti-VEGF therapies are important in thetreatment of certain cancers and in age-related macular degeneration.They can involve monoclonal antibodies such as bevacizumab (AVASTIN),antibody derivatives such as ranibizumab (LUCENTIS), or small moleculesthat inhibit the tyrosine kinases stimulated by VEGF: lapatinib(TYKERB), sunitinib (SUTENT), sorafenib (NEXAVAR), axitinib, andpazopanib. (Some of these therapies target VEGF receptors as opposed tothe VEGFs.)

Some conventional ocular drug delivery systems deliver drugs withtopical eye drops. For example, after cataract and vitreoretinalsurgery, antibiotics are administered dropwise every few hours forseveral days. In addition, other drugs such as non-steroidalanti-inflammatory drugs (NSAIDS) may also need to be given frequently.Some of these eye drops, for example RESTASIS (Allergan), also have astinging and burning sensation associated with their administration.RESTASIS is indicated for dry eye and has to be used by the patientseveral times a day. Similarly treatments for other ophthalmic diseasessuch as cystoid macular edema, diabetic macular edema (DME), anddiabetic retinopathy also need administration of steroidal or NSAIDdrugs. Several vascular proliferative diseases such as maculardegeneration are treated using intravitreal injections of VEGFinhibitors. These include drugs such as LUCENTIS and AVASTIN (Genentech)and MACUGEN (OSI). Such drugs may be delivered using thehydrogel-and-particle systems described herein, with the steps ofrepeated dosings being avoided; e.g., not making new applications of thedrug daily, weekly, or monthly, or not using topical eye drops toadminister the drug.

Various drug delivery systems are known. These various other systemsgenerally include intravitreal implant reservoir type systems,biodegradable depot systems, or implants that need to be removed(non-erodeable). The state of the art in this regard has been delineatedin texts such as “Intraocular Drug Delivery” (Jaffe et al., Taylor &Francis pub., 2006). However, most of these implants either need to beremoved at term, can detach from their target site, may cause visualdisturbances in the back of the eye or can be inflammatory themselvesbecause of the liberation of a substantial amount of acidic degradationproducts. These implants are thus made to be very small with a very highdrug concentration. Even though they are small, they still need to bedeployed with needles over 25 G (25 gauge) in size, or a surgicalapproach delivery system for implantation or removal as needed. Ingeneral, these are localized injections of drug solutions into thevitreous humor or intravitreal implants that use abiodegradable-approach or a removable-reservoir approach. For instance,localized injections delivered into the vitreous humor include anti-VEGFagents LUCENTIS or AVASTIN. POSURDEX (Allergan) is a biodegradableimplant with indications for use being diabetic macular edema (DME) orretinal vein occlusions, with a 22 gauge injector delivery system usedfor delivery into the vitreous cavity; these are powerful drugs in ashort drug delivery duration setting. The therapeutic agent isdexamethasone with polylactic/polyglycolic polymer matrix. Trials withPOSURDEX for diabetic retinopathy are in progress. And for instance, aMEDIDURE implant (PSIVIDA) is used for DME indications. This implant'sthe therapeutic agent is fluocinolone acetonide, and has a nominaldelivery life of 18 months or 36 months (two versions). An intravitreal,removable implant containing triamcinolone acetonide is being tested.Its nominal delivery life is about two years and requires surgicalimplantation. Its indication is for DME.

In contrast to these conventional systems, these or other therapeuticagents may be delivered using a collection of xerogel particles orsystems comprising the particles. The xerogel particles comprise theagent. The xerogels, upon exposure to physiological fluids, imbibe thefluids to form hydrogels that are biocompatible for the eye, which is anenvironment that is distinctly different from other environments. Theuse of minimally inflammatory materials avoids angiogenesis, which isharmful in the eye in many situations. Biocompatible ocular materialsthus avoid unintended angiogenesis; in some aspects, avoiding acidicdegradation products achieves this goal. Further, by using hydrogels andhydrophilic materials (components having a solubility in water of atleast one gram per liter, e.g., polyethylene glycols/oxides), the influxof inflammatory cells is also minimized; this process is in contrast toconventional use of non-hydrogel or rigid, reservoir-based ocularimplants. Moreover, certain proteins may be avoided to enhancebiocompatibility; collagen or fibrin glues, for instance, tend topromote inflammation or unwanted cellular reactions since these releasessignals as they are degraded that promote biological activity. Instead,synthetic materials are used, or peptidic sequences not normally foundin nature. Additionally, biodegradable materials may be used so as toavoid a chronic foreign body reaction, e.g., as with thermally-formedgels that do not degrade. Further, soft materials or materials made insitu to conform the shape of the surrounding tissues can minimize oculardistortion, and low-swelling materials may be used to eliminatevision-distortion caused by swelling. High or low pH materials may beavoided, both in the formation, introduction, or degradation phases.

The xerogels may be prepared with and used to deliver classes of drugs(and drugs to other parts of the body for local as well as systemicdelivery) including steroids, non-steroidal anti-inflammatory drugs(NSAIDS), anti-cancer drugs, antibiotics, or others. The xerogels may beused to deliver drugs and therapeutic agents, e.g., an anti-inflammatory(e.g., Diclofenac), a pain reliever (e.g., Bupivacaine), a Calciumchannel blocker (e.g., Nifedipine), an Antibiotic (e.g., Ciprofloxacin),a Cell cycle inhibitor (e.g., Simvastatin), a protein (e.g., Insulin).The particles may be used to deliver classes of drugs includingsteroids, NSAIDS, antibiotics, pain relievers, inhibitors of vascularendothelial growth factor (VEGF), chemotherapeutics, anti-viral drugs,for instance. Examples of NSAIDS are Ibuprofen, Meclofenamate sodium,mefanamic acid, salsalate, sulindac, tolmetin sodium, ketoprofen,diflunisal, piroxicam, naproxen, etodolac, flurbiprofen, fenoprofencalcium, Indomethacin, celoxib, ketrolac, and nepafenac. The drugsthemselves may be small molecules, proteins, RNA fragments, proteins,glycosaminoglycans, carbohydrates, nucleic acid, inorganic and organicbiologically active compounds where specific biologically active agentsinclude but are not limited to: enzymes, antibiotics, antineoplasticagents, local anesthetics, hormones, angiogenic agents, anti-angiogenicagents, growth factors, antibodies, neurotransmitters, psychoactivedrugs, anticancer drugs, chemotherapeutic drugs, drugs affectingreproductive organs, genes, and oligonucleotides, or otherconfigurations.

A variety of drugs or other therapeutic agents may be delivered usingthese xerogel particles or other xerogel structures. A list of agents orfamilies of drugs and examples of indications for the agents areprovided. The agents may also be used as part of a method of treatingthe indicated condition or making a composition for treating theindicated condition. For example, AZOPT (a brinzolamide opthalmicsuspension) may be used for treatment of elevated intraocular pressurein patients with ocular hypertension or open-angle glaucoma. BETADINE ina Povidone-iodine ophthalmic solution may be used for prepping of theperiocular region and irrigation of the ocular surface. BETOPTIC(betaxolol HCl) may be used to lower intraocular pressure, or forchronic open-angle glaucoma and/or ocular hypertension. CILOXAN(Ciprofloxacin HCl opthalmic solution) may be used to treat infectionscaused by susceptible strains of microorganisms. NATACYN (Natamycinopthalmic suspension) may be used for treatment of fungal blepharitis,conjunctivitis, and keratitis. NEVANAC (Nepanfenac opthalmic suspension)may be used for treatment of pain and inflammation associated withcataract surgery. TRAVATAN (Travoprost ophthalmic solution) may be usedfor reduction of elevated intraocular pressure—open-angle glaucoma orocular hypertension. FML FORTE (Fluorometholone ophthalmic suspension)may be used for treatment of corticosteroid-responsive inflammation ofthe palperbral and bulbar conjunctiva, cornea and anterior segment ofthe globe. LUMIGAN (Bimatoprost ophthalmic solution) may be used forreduction of elevated intraocular pressure—open-angle glaucoma or ocularhypertension. PRED FORTE (Prednisolone acetate) may be used fortreatment of steroid-responsive inflammation of the palpebral and bulbarconjunctiva, cornea and anterior segment of the globe. PROPINE(Dipivefrin hydrochloride) may be used for control of intraocularpressure in chronic open-angle glaucoma. RESTASIS (Cyclosporineophthalmic emulsion) may be used to increases tear production inpatients, e.g., those with ocular inflammation associated withkeratoconjunctivitis sicca. ALREX (Loteprednol etabonate ophthalmicsuspension) may be used for temporary relief of seasonal allergicconjunctivitis. LOTEMAX (Loteprednol etabonate ophthalmic suspension)may be used for treatment of steroid-responsive inflammation of thepalpebral and bulbar conjunctiva, cornea and anterior segment of theglobe. MACUGEN (Pegaptanib sodium injection) may be used for Treatmentof neovascular (wet) age-related macular degeneration. OPTIVAR(Azelastine hydrochloride) may be used for treatment of itching of theeye associated with allergic conjunctivitis. XALATAN (Latanoprostophthalmic solution) may be used to reduce elevated intraocular pressurein patients, e.g., with open-angle glaucoma or ocular hypertension.BETIMOL (Timolol opthalmic solution) may be used for treatment ofelevated intraocular pressure in patients with ocular hypertension oropen-angle glaucoma. Latanoprost is the pro-drug of the free acid form,which is a prostanoid selective FP receptor agonist. Latanoprost reducesintraocular pressure in glaucoma patients with few side effects.Latanoprost has a relatively low solubility in aqueous solutions, but isreadily soluble in organic solvents typically employed for fabricationof microspheres using solvent evaporation.

Further embodiments of agents for delivery include those thatspecifically bind a target peptide in vivo to prevent the interaction ofthe target peptide with its natural receptor or other ligands. AVASTIN,for instance, is an antibody that binds VEGF. And AFLIBERCEPT is afusion protein that includes portions of a VEGF receptor to trap VEGF.An IL-1 trap that makes use of the extracellular domains of IL-1receptors is also known; the trap blocks IL-1 from binding andactivating receptors on the surface of cells. Embodiments of agents fordelivery include nucleic acids, e.g., aptamers. Pegaptanib (MACUGEN),for example, is a pegylated anti-VEGF aptamer. An advantage of theparticle-and-hydrogel delivery process is that the aptamers areprotected from the in vivo environment until they are released. Furtherembodiments of agents for delivery include macromolecular drugs, a termthat refers to drugs that are significantly larger than classical smallmolecule drugs, i.e., drugs such as oligonucleotides (aptamers,antisense, RNAi), ribozymes, gene therapy nucleic acids, recombinantpeptides, and antibodies.

One embodiment comprises extended release of a medication for allergicconjunctivitis. For instance, ketotifen, an antihistamine and mast cellstabilizer, may be provided in particles and released to the eye asdescribed herein in effective amounts to treat allergic conjunctivitis.Seasonal Allergic Conjunctivitis (SAC) and Perennial AllergicConjunctivitis (PAC) are allergic conjunctival disorders. Symptomsinclude itching and pink to reddish eyes. These two eye conditions aremediated by mast cells. Non-specific measures to ameliorate symptomsconventionally include: cold compresses, eyewashes with tearsubstitutes, and avoidance of allergens. Treatment conventionallyconsists of antihistamine mast cell stabilizers, dual mechanismanti-allergen agents, or topical antihistamines. Corticosteroids mightbe effective but, because of side effects, are reserved for more severeforms of allergic conjunctivitis such as vernal keratoconjunctivitis(VKC) and atopic keratoconjunctivitis (AKC).

Moxifloxacin is the active ingredient in VIGAMOX, which is afluoroquinolone approved for use to treat or prevent ophthalmicbacterial infections. Dosage is typically one-drop of a 0.5% solutionthat is administered 3 times a day for a period of one-week or more.

VKC and AKC are chronic allergic diseases where eosinophils,conjunctival fibroblasts, epithelial cells, mast cells, and/or TH2lymphocytes aggravate the biochemistry and histology of the conjunctiva.VKC and AKC can be treated by medications used to combat allergicconjunctivitis.

Permeation agents are agents and may also be included in a gel,hydrogel, organogel, xerogel, and biomaterials as described herein.These are agents that assist in permeation of a drug into an intendedtissue. Permeation agents may be chosen as needed for the tissue, e.g.,permeation agents for skin, permeation agents for an eardrum, permeationagents for an eye.

Xerogel Particle Blending and Collections

A collection of particles (powder particles of an agent and/orxerogel/hydrogel particles) may include sets of particles. The termxerogel/hydrogel refers to xerogels and/or thexerogels-hydrated-as-hydrogels. For instance, a collection may includesome xerogel particles that contain a radioopaque agent, with thoseparticles forming a set within the collection. Other sets are directedto particle sizes, with the sets having distinct shapes or sizedistributions. As discussed, particles can be made with well-controlledsizes and can thus be made and divided into various sets for combinationinto a collection.

Some sets are made of particles (xerogel/hydrogel) with a particulardegradability. One embodiment involves a plurality of sets each having adistinct degradability profile. The different degradation rates providedifferent release profiles. Combinations of the different sets ofparticles may be made to achieve a desired profile, as demonstrated inFIGS. 6 and 7, referring to Example 2. Degradation times include 3 to1000 days; artisans will immediately appreciate that all the ranges andvalues within the explicitly stated ranges are contemplated. Forinstance, a first set may have a median degradation time of from about 5to about 8 days, a second set a median time of from about 30 to about 90days, and a third set a median time of from about 180 to about 360 days.

Xerogel/hydrogel particles may be blended to achieve a desired proteinrelease profile. Gels with different degradation rates (as hydrogels)can be combined to provide constant or near constant release thatcompensates for the inherently non-linear release profile of singlegels.

A collection of xerogel/hydrogel particles may include sets of agents.For instance, some particles may be made to contain a first therapeuticagent, with those particles forming a set within the collection. Andother sets may have another agent. Examples of agents are water solublebiologics, proteins, peptides, nucleic acids, small molecule drugs, andhydrophobic agents. Other sets may be directed to particle sizes, withthe sets having distinct shapes or size distributions. As discussed,particles can be made with well-controlled sizes and divided intovarious sets for combination into a collection. These various sets maybe freely mixed-and-matched in combinations and subcombinations, forexample: sizes, degradability, therapeutic agents, and visualizationagents.

Xerogel/hydrogels may further comprise agents that are not in a powderform. The agents may be disposed with the xerogel/hydrogel or mixed withthe solution of other vehicle that is used with the xerogel/hydrogel.For example, a collection of xerogel particles may be hydrated at pointof use to form a hydrogel by adding water or saline that furthercomprises a drug solution. Such drugs or agents may be the same as theagent that is in a powder in the xerogel/hydrogel so as to provide aninitial burst of release, or may be for secondary therapy orvisualization.

Lubricity

Collections may be made with sizes and lubricity for manual injectionthrough a small gauge needle. Hydrophilic hydrogels crushed intospheroidal particles about 40 to about 100 microns diameter are smallenough to be manually injected through a 30 gauge needle. Hydrophilichydrogel particles were observed to pass with difficulty through smallgauge needles/catheters, as reported in U.S. Publication No.2011/0142936, which is hereby incorporated herein for all purposes; incase of conflict, the instant specification is controlling. The particlesize contributes to resistance, as well as the viscosity of thesolution. The particles tended to plug the needle. The resistance forceis proportional to the viscosity of the fluid, with a more viscous fluidrequiring more force to push through a small opening.

As reported in U.S. Publication No. 2011/0142936, it was unexpectedlyfound that increasing the viscosity of the solvent for the particlescould lower the resistance to passage through a catheter and/or needle.This decrease may be attributed to using a solvent with a highosmolarity. Without being bound to a particular theory, the addition ofthese agents to improve injectability was caused by particle shrinkage,increased free water between particles which decreasedparticle-to-particle contributions to viscosity, and increased viscosityof the free water, which helped to pull the particles into and out ofthe syringes, preventing straining and plugging. The use of a linearpolymer may further contribute thixotropic properties that are useful toprevent settling and encourage movement of the particles together withthe solvent, but exhibit shear thinning when being forced out of a smallopening. This approach was also observed to solve another problem,namely, a difficulty in moving particles from a solution through aneedle/catheter since the particles tended to settle and otherwise eludepick-up. Expulsion through small bore openings of solutions of particlesin aqueous solvent were observed; the solvent tended to movepreferentially out of the applicator, leaving an excess of particlesbehind that could not be cleared from the applicator, or that pluggedit, or in some instances could be cleared but only by use of anunsuitably large force not suited to an average user operating ahand-held syringe. The addition of osmotic agents, however, contributedviscosity and/or thixotropic behavior that helped to empty particlesfrom an applicator.

Embodiments of the invention include the addition of an osmotic agent toa plurality of xerogel/hydrogel particles. Examples of such agentsinclude salts and polymers. Embodiments include polymers, linearpolymers, and hydrophilic polymers, or combinations of the same.Embodiments include polymers of between about 500 and about 100,000molecular weight; artisans will immediately appreciate that all theranges and values within the explicitly stated ranges are contemplated,e.g., about 5000 to about 50,000 molecular weight. Embodiments include,for example, a concentration of about 1% to about 50% w/w osmotic agent;artisans will immediately appreciate that all the ranges and valueswithin the explicitly stated ranges are contemplated, e.g., 10% to 30%.The agent and hydrogel may be introduced into a patient and may be partof a kit for the same.

Precursors

Matrices may be prepared and used to contain the particles of watersoluble biologics. Accordingly, embodiments are provided herein formaking implantable matrices. Such matrices include matrices with aporosity of more than about 20% v/v; artisans will immediatelyappreciate that all the ranges and values within the explicitly statedrange is contemplated. Precursors may be dissolved in an organic solventto make an organogel. An organogel is a non-crystalline, non-glassysolid material composed of a liquid organic phase entrapped in athree-dimensionally cross-linked network. The liquid can be, forexample, an organic solvent, mineral oil, or vegetable oil. Thesolubility and dimensions of the solvent are important characteristicsfor the elastic properties and firmness of the organogel. Alternatively,the precursor molecules may themselves be capable of forming their ownorganic matrix, eliminating the need for a tertiary organic solvent. Theterm precursor refers to a component that becomes part of thecrosslinked matrix. A polymer that becomes crosslinked into the matrixis a precursor while a salt or a protein that is merely present in thematrix is not a precursor.

Removal of the solvent (if used) from the organogel provides a xerogel,a dried gel. The xerogels formed by, for example, freeze drying, mayhave a high porosity (at least about 20%, a large surface area, and asmall pore size. Xerogels made with hydrophilic materials form hydrogelswhen exposed to aqueous solutions. High porosity xerogels hydrate morequickly than more dense xerogels. Hydrogels are materials that do notdissolve in water and retain a significant fraction (more than 20%) ofwater within their structure. In fact, water contents in excess of 90%are often known. Hydrogels may be formed by crosslinking water solublemolecules to form networks of essentially infinite molecular weight.Hydrogels with high water contents are typically soft, pliablematerials. Hydrogels and drug delivery systems as described in U.S.Publication Nos. 2009/0017097, 2011/0142936 and 2012/0071865 may beadapted for use with the materials and methods herein by following theguidance provided herein; these references are hereby incorporatedherein by reference for all purposes, and in case of conflict, theinstant specification is controlling.

Organogels and hydrogels may be formed from natural, synthetic, orbiosynthetic polymers. Natural polymers may include glycosaminoglycans,polysaccharides, and proteins. Some examples of glycosaminoglycansinclude dermatan sulfate, hyaluronic acid, the chondroitin sulfates,chitin, heparin, keratan sulfate, keratosulfate, and derivativesthereof. In general, the glycosaminoglycans are extracted from a naturalsource and purified and derivatized. However, they also may besynthetically produced or synthesized by modified microorganisms such asbacteria. These materials may be modified synthetically from a naturallysoluble state to a partially soluble or water swellable or hydrogelstate. This modification may be accomplished by various well-knowntechniques, such as by conjugation or replacement of ionizable orhydrogen bondable functional groups such as carboxyl and/or hydroxyl oramine groups with other more hydrophobic groups.

For example, carboxyl groups on hyaluronic acid may be esterified byalcohols to decrease the solubility of the hyaluronic acid. Suchprocesses are used by various manufacturers of hyaluronic acid products(such as Genzyme Corp., Cambridge, Mass.) to create hyaluronic acidbased sheets, fibers, and fabrics that form hydrogels. Other naturalpolysaccharides, such as carboxymethyl cellulose or oxidized regeneratedcellulose, natural gum, agar, agrose, sodium alginate, carrageenan,fucoidan, furcellaran, laminaran, hypnea, eucheuma, gum arabic, gumghatti, gum karaya, gum tragacanth, locust beam gum, arbinoglactan,pectin, amylopectin, gelatin, hydrophilic colloids such as carboxymethylcellulose gum or alginate gum crosslinked with a polyol such aspropylene glycol, and the like, also form hydrogels upon contact withaqueous surroundings.

Synthetic organogels or hydrogels may be biostable or biodegradable.Examples of biostable hydrophilic polymeric materials arepoly(hydroxyalkyl methacrylate), poly(electrolyte complexes),poly(vinylacetate) cross-linked with hydrolysable or otherwisedegradable bonds, and water-swellable N-vinyl lactams. Other hydrogelsinclude hydrophilic hydrogels known as CARBOPOL®, an acidic carboxypolymer (Carbomer resins are high molecular weight,allylpentaerythritol-crosslinked, acrylic acid-based polymers, modifiedwith C10-C30 alkyl acrylates), polyacrylamides, polyacrylic acid, starchgraft copolymers, acrylate polymer, ester cross-linked polyglucan. Suchhydrogels are described, for example, in U.S. Pat. No. 3,640,741 toEtes, U.S. Pat. No. 3,865,108 to Hartop, U.S. Pat. No. 3,992,562 toDenzinger et al., U.S. Pat. No. 4,002,173 to Manning et al., U.S. Pat.No. 4,014,335 to Arnold and U.S. Pat. No. 4,207,893 to Michaels, all ofwhich are incorporated herein by reference, with the presentspecification controlling in case of conflict.

Hydrogels and organogels may be made from precursors. The precursors arenot the hydrogels/organogels but are crosslinked with each other to formthe hydrogel/organogel. Crosslinks can be formed by covalent bonds orphysical bonds. Examples of physical bonds are ionic bonds, hydrophobicassociation of precursor molecule segments, and crystallization ofprecursor molecule segments. The precursors can be triggered to react toform a crosslinked hydrogel. The precursors can be polymerizable andinclude crosslinkers that are often, but not always, polymerizableprecursors. Polymerizable precursors are thus precursors that havefunctional groups that react with each other to form matrices and/orpolymers made of repeating units. Precursors may be polymers.

Some precursors thus react by chain-growth polymerization, also referredto as addition polymerization, and involve the linking together ofmonomers incorporating double or triple chemical bonds. Theseunsaturated monomers have extra internal bonds which are able to breakand link up with other monomers to form the repeating chain. Monomersare polymerizable molecules with at least one group that reacts withother groups to form a polymer. A macromonomer (or macromer) is apolymer or oligomer that has at least one reactive group, often at theend, which enables it to act as a monomer; each macromonomer molecule isattached to the polymer by reaction the reactive group. Thusmacromonomers with two or more monomers or other functional groups tendto form covalent crosslinks. Addition polymerization is involved in themanufacture of e.g., polypropylene or polyvinyl chloride. One type ofaddition polymerization is living polymerization.

Some precursors thus react by condensation polymerization that occurswhen monomers bond together through condensation reactions. Typicallythese reactions can be achieved through reacting molecules incorporatingalcohol, amine or carboxylic acid (or other carboxyl derivative)functional groups. When an amine reacts with a carboxylic acid an amideor peptide bond is formed, with the release of water. Some condensationreactions follow a nucleophilic acyl substitution, e.g., as in U.S. Pat.No. 6,958,212, which is hereby incorporated by reference herein in itsentirety to the extent it does not contradict what is explicitlydisclosed herein.

Some precursors react by a chain growth mechanism. Chain growth polymersare defined as polymers formed by the reaction of monomers ormacromonomers with a reactive center. A reactive center is a particularlocation within a chemical compound that is the initiator of a reactionin which the chemical is involved. In chain-growth polymer chemistry,this is also the point of propagation for a growing chain. The reactivecenter is commonly radical, anionic, or cationic in nature, but can alsotake other forms. Chain growth systems include free radicalpolymerization, which involves a process of initiation, propagation andtermination. Initiation is the creation of free radicals necessary forpropagation, as created from radical initiators, e.g., organic peroxidemolecules. Termination occurs when a radical reacts in a way thatprevents further propagation. The most common method of termination isby coupling where two radical species react with each other forming asingle molecule.

Some precursors react by a step growth mechanism, and are polymersformed by the stepwise reaction between functional groups of monomers.Most step growth polymers are also classified as condensation polymers,but not all step growth polymers release condensates.

Monomers may be polymers or small molecules. A polymer is a highmolecular weight molecule formed by combining many smaller molecules(monomers) in a regular pattern. Oligomers are polymers having less thanabout 20 monomeric repeat units. A small molecule generally refers to amolecule that is less than about 2000 Daltons.

The precursors may thus be small molecules, such as acrylic acid orvinyl caprolactam, larger molecules containing polymerizable groups,such as acrylate-capped polyethylene glycol (PEG-diacrylate), or otherpolymers containing ethylenically-unsaturated groups, such as those ofU.S. Pat. No. 4,938,763 to Dunn et al, U.S. Pat. Nos. 5,100,992 and4,826,945 to Cohn et al, or U.S. Pat. Nos. 4,741,872 and 5,160,745 toDeLuca et al., each of which is hereby incorporated by reference hereinin its entirety to the extent it does not contradict what is explicitlydisclosed herein.

To form covalently crosslinked hydrogels, the precursors must becovalently crosslinked together. In general, polymeric precursors arepolymers that will be joined to other polymeric precursors at two ormore points, with each point being a linkage to the same or differentpolymers. Precursors with at least two reactive centers (for example, infree radical polymerization) can serve as crosslinkers since eachreactive group can participate in the formation of a different growingpolymer chain. In the case of functional groups without a reactivecenter, among others, crosslinking requires three or more suchfunctional groups on at least one of the precursor types. For instance,many electrophilic-nucleophilic reactions consume the electrophilic andnucleophilic functional groups so that a third functional group isneeded for the precursor to form a crosslink. Such precursors thus mayhave three or more functional groups and may be crosslinked byprecursors with two or more functional groups. A crosslinked moleculemay be crosslinked via an ionic or covalent bond, a physical force, orother attraction. A covalent crosslink, however, will typically offerstability and predictability in reactant product architecture.

In some embodiments, each precursor is multifunctional, meaning that itcomprises two or more electrophilic or nucleophilic functional groups,such that a nucleophilic functional group on one precursor may reactwith an electrophilic functional group on another precursor to form acovalent bond. At least one of the precursors comprises more than twofunctional groups, so that, as a result of electrophilic-nucleophilicreactions, the precursors combine to form crosslinked polymericproducts.

The precursors may have biologically inert and hydrophilic portions,e.g., a core. In the case of a branched polymer, a core refers to acontiguous portion of a molecule joined to arms that extend from thecore, with the anus having a functional group, which is often at theterminus of the branch. A hydrophilic precursor or precursor portion hasa solubility of at least 1 g/100 mL in an aqueous solution. Ahydrophilic portion may be, for instance, a polyether, for example,polyalkylene oxides such as polyethylene glycol (PEG), polyethyleneoxide (PEO), polyethylene oxide-co-polypropylene oxide (PPO),co-polyethylene oxide block or random copolymers, and polyvinyl alcohol(PVA), poly(vinyl pyrrolidinone) (PVP), poly(amino acids, dextran, or aprotein. The precursors may have a polyalkylene glycol portion and maybe polyethylene glycol based, with at least about 80% or 90% by weightof the polymer comprising polyethylene oxide repeats. The polyethers andmore particularly poly(oxyalkylenes) or poly(ethylene glycol) orpolyethylene glycol are generally hydrophilic. As is customary in thesearts, the term PEG is used to refer to PEO with or without hydroxyl endgroups.

A precursor may also be a macromolecule (or macromer), which is amolecule having a molecular weight in the range of a thousand to manymillions. In some embodiments, however, at least one of the precursorsis a small molecule of about 1000 Da or less. The macromolecule, whenreacted in combination with a small molecule of about 1000 Da or less,is preferably at least five to fifty times greater in molecular weightthan the small molecule and is preferably less than about 60,000 Da;artisans will immediately appreciate that all the ranges and valueswithin the explicitly stated ranges are contemplated. A more preferredrange is a macromolecule that is about seven to about thirty timesgreater in molecular weight than the crosslinker and a most preferredrange is about ten to twenty times difference in weight. Further, amacromolecular molecular weight of 5,000 to 50,000 is useful, as is amolecular weight of 7,000 to 40,000 or a molecular weight of 10,000 to20,000.

Certain macromeric precursors are the crosslinkable, biodegradable,water-soluble macromers described in U.S. Pat. No. 5,410,016 to Hubbellet al, which is hereby incorporated herein by reference in its entiretyto the extent it does not contradict what is explicitly disclosed. Thesemacromers are characterized by having at least two polymerizable groups,separated by at least one degradable region.

Synthetic precursors may be used. Synthetic refers to a molecule notfound in nature or not normally found in a human. Some syntheticprecursors are free of amino acids or free of amino acid sequences thatoccur in nature. Some synthetic precursors are polypeptides that are notfound in nature or are not normally found in a human body, e.g., di-,tri-, or tetra-lysine. Some synthetic molecules have amino acid residuesbut only have one, two, or three that are contiguous, with the aminoacids or clusters thereof being separated by non-natural polymers orgroups. Polysaccharides or their derivatives are thus not synthetic.

Alternatively, natural proteins or polysaccharides may be adapted foruse with these methods, e.g., collagens, fibrin(ogen)s, albumins,alginates, hyaluronic acid, and heparins. These natural molecules mayfurther include chemical derivitization, e.g., synthetic polymerdecorations. The natural molecule may be crosslinked via its nativenucleophiles or after it is derivatized with functional groups, e.g., asin U.S. Pat. Nos. 5,304,595, 5,324,775, 6,371,975, and 7,129,210, eachof which is hereby incorporated by reference to the extent it does notcontradict what is explicitly disclosed herein. Natural refers to amolecule found in nature. Natural polymers, for example proteins orglycosaminoglycans, e.g., collagen, fibrinogen, albumin, and fibrin, maybe crosslinked using reactive precursor species with electrophilicfunctional groups. Natural polymers normally found in the body areproteolytically degraded by proteases present in the body. Such polymersmay be reacted via functional groups such as amines, thiols, orcarboxyls on their amino acids or derivatized to have activatablefunctional groups. While natural polymers may be used in hydrogels,their time to gelation and ultimate mechanical properties must becontrolled by appropriate introduction of additional functional groupsand selection of suitable reaction conditions, e.g., pH.

Precursors may be made with a hydrophobic portion provided that theresultant hydrogel retains the requisite amount of water, e.g., at leastabout 20%. In some cases, the precursor is nonetheless soluble in waterbecause it also has a hydrophilic portion. In other cases, the precursormakes dispersion in the water (a suspension) but is nonethelessreactable to from a crosslinked material. Some hydrophobic portions mayinclude a plurality of alkyls, polypropylenes, alkyl chains, or othergroups. Some precursors with hydrophobic portions are sold under thetrade names PLURONIC F68, JEFFAMINE, or TECTRONIC. A hydrophobicmolecule or a hydrophobic portion of a copolymer or the like is one thatis sufficiently hydrophobic to cause the molecule (e.g., polymer orcopolymer) to aggregate to form micelles or microphases involving thehydrophobic domains in an aqueous continuous phase or one that, whentested by itself, is sufficiently hydrophobic to precipitate from, orotherwise change phase while within, an aqueous solution of water at pHfrom about 7 to about 7.5 at temperatures from about 30 to about 50degrees Centigrade.

Precursors may have, e.g., 2-100 arms, with each arm having a terminus,bearing in mind that some precursors may be dendrimers or other highlybranched materials. An arm on a hydrogel precursor refers to a linearchain of chemical groups that connect a crosslinkable functional groupto a polymer core. Some embodiments are precursors with between 3 and300 arms; artisans will immediately appreciate that all the ranges andvalues within the explicitly stated ranges are contemplated, e.g., 4 to16, 8 to 100, or at least 6 arms.

Thus hydrogels can be made, e.g., from a multi-armed precursor with afirst set of functional groups and a low molecular-weight precursorhaving a second set of functional groups. For example, a six-armed oreight-armed precursor may have hydrophilic arms, e.g., polyethyleneglycol, terminated with primary amines, with the molecular weight of thearms being about 1,000 to about 40,000; artisans will immediatelyappreciate that all ranges and values within the explicitly statedbounds are contemplated. Such precursors may be mixed with relativelysmaller precursors, for example, molecules with a molecular weight ofbetween about 100 and about 5000, or no more than about 800, 1000, 2000,or 5000 having at least about three functional groups, or between about3 to about 16 functional groups; ordinary artisans will appreciate thatall ranges and values between these explicitly articulated values arecontemplated. Such small molecules may be polymers or non-polymers andnatural or synthetic.

Precursors that are not dendrimers may be used. Dendritic molecules arehighly branched radially symmetrical polymers in which the atoms arearranged in many arms and subarms radiating out from a central core.Dendrimers are characterized by their degree of structural perfection asbased on the evaluation of both symmetry and polydispersity and requireparticular chemical processes to synthesize. Accordingly, an artisan canreadily distinguish dendrimer precursors from non-dendrimer precursors.Dendrimers have a shape that is typically dependent on the solubility ofits component polymers in a given environment, and can changesubstantially according to the solvent or solutes around it, e.g.,changes in temperature, pH, or ion content.

Precursors may be dendrimers, e.g., as in U.S. Publication Nos.2004/0086479 and 2004/0131582 and PCT Publication Nos. WO07005249,WO07001926 and WO06031358, or the U.S. counterparts thereof; dendrimersmay also be useful as multifunctional precursors, e.g., as in U.S.Publication Nos. 2004/0131582 and 2004/0086479 and PCT Publication Nos.WO06031388 and WO06031388; each of which US and PCT applications arehereby incorporated by reference herein in its entirety to the extentthey do not contradict what is explicitly disclosed herein. Dendrimersare highly ordered possess high surface area to volume ratios, andexhibit numerous end groups for potential functionalization. Embodimentsinclude multifunctional precursors that are not dendrimers.

Some embodiments include a precursor that consists essentially of anoligopeptide sequence of no more than five residues, e.g., amino acidscomprising at least one amine, thiol, carboxyl, or hydroxyl side chain.A residue is an amino acid, either as occurring in nature or derivatizedthereof. The backbone of such an oligopeptide may be natural orsynthetic. In some embodiments, peptides of two or more amino acids arecombined with a synthetic backbone to make a precursor; certainembodiments of such precursors have a molecular weight in the range ofabout 100 to about 10,000 or about 300 to about 500 Artisans willimmediately appreciate that all ranges and values between theseexplicitly articulated bounds are contemplated.

Precursors may be prepared to be free of amino acid sequences cleavableby enzymes present at the site of introduction, including free ofsequences susceptible to attach by metalloproteinases and/orcollagenases. Further, precursors may be made to be free of all aminoacids, or free of amino acid sequences of more than about 50, 30, 20,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids. Precursors may benon-proteins, meaning that they are not a naturally occurring proteinand can not be made by cleaving a naturally occurring protein and cannot be made by adding synthetic materials to a protein. Precursors maybe non-collagen, non-fibrin, non-fibrinogen, and non-albumin, meaningthat they are not one of these proteins and are not chemical derivativesof one of these proteins. The use of non-protein precursors and limiteduse of amino acid sequences can be helpful for avoiding immunereactions, avoiding unwanted cell recognition, and avoiding the hazardsassociated with using proteins derived from natural sources. Precursorscan also be non-saccharides (free of saccharides) or essentiallynon-saccharides (free of more than about 5% saccharides by w/w of theprecursor molecular weight. Thus a precursor may, for example, excludehyaluronic acid, heparin, or gellan. Precursors can also be bothnon-proteins and non-saccharides.

Peptides may be used as precursors. In general, peptides with less thanabout 10 residues are preferred, although larger sequences (e.g.,proteins) may be used. Artisans will immediately appreciate that everyrange and value within these explicit bounds is included, e.g., 1-10,2-9, 3-10, 1, 2, 3, 4, 5, 6, or 7. Some amino acids have nucleophilicgroups (e.g., primary amines or thiols) or groups that can bederivatized as needed to incorporate nucleophilic groups orelectrophilic groups (e.g., carboxyls or hydroxyls). Polyamino acidpolymers generated synthetically are normally considered to be syntheticif they are not found in nature and are engineered not to be identicalto naturally occurring biomolecules.

Some organogels and hydrogels are made with a polyethyleneglycol-containing precursor. Polyethylene glycol (PEG, also referred toas polyethylene oxide when occurring in a high molecular weight) refersto a polymer with a repeat group (CH₂CH₂O)_(n), with n being at least 3.A polymeric precursor having a polyethylene glycol thus has at leastthree of these repeat groups connected to each other in a linear series.The polyethylene glycol content of a polymer or arm is calculated byadding up all of the polyethylene glycol groups on the polymer or arm,even if they are interrupted by other groups. Thus, an arm having atleast 1000 MW polyethylene glycol has enough CH₂CH₂O groups to total atleast 1000 MW. As is customary terminology in these arts, a polyethyleneglycol polymer does not necessarily refer to a molecule that terminatesin a hydroxyl group. Molecular weights are abbreviated in thousandsusing the symbol k, e.g., with 15K meaning 15,000 molecular weight,i.e., 15,000 Daltons. SG refers to succinimidyl glutarate. SS refers tosuccinimidyl succinate. SAP refers to succinimidyl adipate. SAZ refersto succinimidyl azelate. SS, SG, SAP and SAZ are succinimidyl estersthat have an ester group that degrades by hydrolysis in water.Hydrolytically degradable thus refers to a material that wouldspontaneously degrade in vitro in an excess of water without any enzymesor cells present to mediate the degradation. A time for degradationrefers to effective disappearance of the material as judged by the nakedeye. Trilysine (also abbreviated LLL) is a synthetic tripeptide. PEGand/or hydrogels, as well as compositions that comprise the same, may beprovided in a form that is pharmaceutically acceptable, meaning that itis highly purified and free of contaminants, e.g., pyrogens.

Functional Groups

The precursors for covalent crosslinking have functional groups thatreact with each other to form the material, either outside a patient, orin situ. The functional groups generally have polymerizable groups forpolymerization or react with each other in electrophile-nucleophilereactions or are configured to participate in other polymerizationreactions. Various aspects of polymerization reactions are discussed inthe precursors section herein.

Thus in some embodiments, precursors have a polymerizable group that isactivated by photoinitiation or redox systems as used in thepolymerization arts, e.g., or electrophilic functional groups that arecarbodiimidazole, sulfonyl chloride, chlorocarbonates,n-hydroxysucciniinidyl ester, succinimidyl ester or sulfasuccinimidylesters, or as in U.S. Pat. Nos. 5,410,016 or 6,149,931, each of whichare hereby incorporated by reference herein in its entirety to theextent they do not contradict what is explicitly disclosed herein. Thenucleophilic functional groups may be, for example, amine, hydroxyl,carboxyl, and thiol. Another class of electrophiles are acyls, e.g., asin U.S. Pat. No. 6,958,212, which describes, among other things, Michaeladdition schemes for reacting polymers.

Certain functional groups, such as alcohols or carboxylic acids, do notnormally react with other functional groups, such as amines, underphysiological conditions (e.g., pH 7.2-11.0, 37° C.). However, suchfunctional groups can be made more reactive by using an activating groupsuch as N-hydroxysuccinimide. Certain activating groups includecarbonyldiimidazole, sulfonyl chloride, aryl halides, sulfasuccinimidylesters, N-hydroxysuccinimidyl ester, succinimidyl ester, epoxide,aldehyde, maleimides, imidoesters and the like. The N-hydroxysuccinimideesters or N-hydroxysulfosuccinimide (NHS) groups are useful groups forcrosslinking of proteins or amine-containing polymers, e.g., aminoterminated polyethylene glycol. An advantage of an NHS-amine reaction isthat the reaction kinetics are favorable, but the gelation rate may beadjusted through pH or concentration. The NHS-amine crosslinkingreaction leads to formation of N-hydroxysuccinimide as a side product.Sulfonated or ethoxylated forms of N-hydroxysuccinimide have arelatively increased solubility in water and hence their rapid clearancefrom the body. An NHS-amine crosslinking reaction may be carried out inaqueous solutions and in the presence of buffers, e.g., phosphate buffer(pH 5.0-7.5), triethanolamine buffer (pH 7.5-9.0), or borate buffer (pH9.0-12), or sodium bicarbonate buffer (pH 9.0-10.0). Aqueous solutionsof NHS based crosslinkers and functional polymers preferably are madejust before the crosslinking reaction due to reaction of NHS groups withwater. The reaction rate of these groups may be delayed by keeping thesesolutions at lower pH (pH 4-7). Buffers may also be included in thehydrogels introduced into a body.

In some embodiments, each precursor comprises only nucleophilic or onlyelectrophilic functional groups, so long as both nucleophilic andelectrophilic precursors are used in the crosslinking reaction. Thus,for example, if a crosslinker has nucleophilic functional groups such asamines, the functional polymer may have electrophilic functional groupssuch as N-hydroxysuccinimides. On the other hand, if a crosslinker haselectrophilic functional groups such as sulfosuccinimides, then thefunctional polymer may have nucleophilic functional groups such asamines or thiols. Thus, functional polymers such as proteins, poly(allylamine), or amine-terminated di- or multifunctional poly(ethylene glycol)can be used.

One embodiment has reactive precursor species with 3 to 16 nucleophilicfunctional groups each and reactive precursor species with 2 to 12electrophilic functional groups each; artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated.

The functional groups may be, e.g., electrophiles reactable withnucleophiles, groups reactable with specific nucleophiles, e.g., primaryamines, groups that form amide bonds with materials in the biologicalfluids, groups that form amide bonds with carboxyls, activated-acidfunctional groups, or a combination of the same. The functional groupsmay be, e.g., a strong electrophilic functional group, meaning anelectrophilic functional group that effectively forms a covalent bondwith a primary amine in aqueous solution at pH 9.0 at room temperatureand pressure and/or an electrophilic group that reacts by a ofMichael-type reaction. The strong electrophile may be of a type thatdoes not participate in a Michaels-type reaction or of a type thatparticipates in a Michaels-type reaction.

A Michael-type reaction refers to the 1,4 addition reaction of anucleophile on a conjugate unsaturated system. The addition mechanismcould be purely polar, or proceed through a radical-like intermediatestate(s); Lewis acids or appropriately designed hydrogen bonding speciescan act as catalysts. The term conjugation can refer both to alternationof carbon-carbon, carbon-heteroatom or heteroatom-heteroatom multiplebonds with single bonds, or to the linking of a functional group to amacromolecule, such as a synthetic polymer or a protein. Michael-typereactions are discussed in detail in U.S. Pat. No. 6,958,212, which ishereby incorporated by reference herein in its entirety for all purposesto the extent it does not contradict what is explicitly disclosedherein.

Examples of strong electrophiles that do not participate in aMichaels-type reaction are: succinimides, succinimidyl esters, orNHS-esters. Examples of Michael-type electrophiles are acrylates,methacrylates, methylmethacrylates, and other unsaturated polymerizablegroups.

Initiating Systems

Some precursors react using initiators. An initiator group is a chemicalgroup capable of initiating a free radical polymerization reaction. Forinstance, it may be present as a separate component, or as a pendentgroup on a precursor. Initiator groups include thermal initiators,photoactivatable initiators, and oxidation-reduction (redox) systems.Long wave UV and visible light photoactivatable initiators include, forexample, ethyl eosin groups, 2,2-dimethoxy-2-phenyl acetophenone groups,other acetophenone derivatives, thioxanthone groups, benzophenonegroups, and camphorquinone groups. Examples of thermally reactiveinitiators include 4,4′ azobis(4-cyanopentanoic acid) groups, andanalogs of benzoyl peroxide groups. Several commercially available lowtemperature free radical initiators, such as V-044, available from WakoChemicals USA, Inc., Richmond, Va., may be used to initiate free radicalcrosslinking reactions at body temperatures to form hydrogel coatingswith the aforementioned monomers.

Metal ions may be used either as an oxidizer or a reductant in redoxinitiating systems. For example, ferrous ions may be used in combinationwith a peroxide or hydroperoxide to initiate polymerization, or as partsof a polymerization system. In this case, the ferrous ions would serveas a reductant. Alternatively, metal ions may serve as an oxidant. Forexample, the ceric ion (4+ valence state of cerium) interacts withvarious organic groups, including carboxylic acids and urethanes, toremove an electron to the metal ion, and leave an initiating radicalbehind on the organic group. In such a system, the metal ion acts as anoxidizer. Potentially suitable metal ions for either role are any of thetransition metal ions, lanthanides and actinides, which have at leasttwo readily accessible oxidation states. Particularly useful metal ionshave at least two states separated by only one difference in charge. Ofthese, the most commonly used are ferric/ferrous; cupric/cuprous;ceric/cerous; cobaltic/cobaltous; vanadate V vs. IV; permanganate; andmanganic/manganous. Peroxygen containing compounds, such as peroxidesand hydroperoxides, including hydrogen peroxide, t-butyl hydroperoxide,t-butyl peroxide, benzoyl peroxide, cumyl peroxide may be used.

An example of an initiating system is the combination of a peroxygencompound in one solution, and a reactive ion, such as a transitionmetal, in another. In this case, no external initiators ofpolymerization are needed and polymerization proceeds spontaneously andwithout application of external energy or use of an external energysource when two complementary reactive functional groups containingmoieties interact at the application site.

Visualization Agents

A visualization agent may be used as a powder in a xerogel/hydrogel; itreflects or emits light at a wavelength detectable to a human eye sothat a user applying the hydrogel could observe the object when itcontains an effective amount of the agent. Agents that require a machineaid for imaging are referred to as imaging agents herein, and examplesinclude: radioopaque contrast agents and ultrasound contrast agents.

Some biocompatible visualization agents are FD&C BLUE #1, FD&C BLUE #2,and methylene blue. These agents are preferably present in the finalelectrophilic-nucleophilic reactive precursor species mix at aconcentration of more than 0.05 mg/ml and preferably in a concentrationrange of at least 0.1 to about 12 mg/ml, and more preferably in therange of 0.1 to 4.0 mg/ml, although greater concentrations maypotentially be used, up to the limit of solubility of the visualizationagent. Visualization agents may be covalently linked to the molecularnetwork of the xerogel/hydrogel, thus preserving visualization afterapplication to a patient until the hydrogel hydrolyzes to dissolution.

Visualization agents may be selected from among any of the variousnon-toxic colored substances suitable for use in medical implantablemedical devices, such as FD&C BLUE dyes 3 and 6, eosin, methylene blue,indocyanine green, or colored dyes normally found in synthetic surgicalsutures. Reactive visualization agents such as NHS-fluorescein can beused to incorporate the visualization agent into the molecular networkof the xerogel/hydrogel. The visualization agent may be present witheither reactive precursor species, e.g., a crosslinker or functionalpolymer solution. The preferred colored substance may or may not becomechemically bound to the hydrogel. The visualization agent may be used insmall quantities, e.g., 1% weight/volume, more preferably less that0.01% weight/volume and most preferably less than 0.001% weight/volumeconcentration; artisans will immediately appreciate that all the rangesand values within the explicitly stated ranges are contemplated. Theagent tends to mark the location of the particle and provides anindication of its presence and dissolution rate.

Biodegradation

The xerogel may be formed from the organogel so that, upon hydration inphysiological solution, a hydrogel is formed that is water-degradable,as measurable by the hydrogel losing its mechanical strength andeventually dissipating in vitro in an excess of water by hydrolyticdegradation of water-degradable groups. This test is predictive ofhydrolytically-driven dissolution in vivo, a process that is in contrastto cell or protease-driven degradation. Significantly, however,polyanhydrides or other conventionally-used degradable materials thatdegrade to acidic components tend to cause inflammation in tissues. Thehydrogels, however, may exclude such materials, and may be free ofpolyanhydrides, anhydride bonds, or precursors that degrade into acid ordiacids. The term degradation by solvation in water, also referred to asdissolving in water, refers to a process of a matrix gradually goinginto solution in, which is a process that can not take place for acovalently crosslinked material and materials insoluble in water.

For example, electrophilic groups such as SG (N-hydroxysuccinimidylglutarate), SS (N-hydroxysuccinimidyl succinate), SC(N-hydroxysuccinimidyl carbonate), SAP (N-hydroxysuccinimidyl adipate)or SAZ (N-hydroxysuccinimidyl azelate) may be used and have estericlinkages that are hydrolytically labile. More linear hydrophobiclinkages such as pimelate, suberate, azelate or sebacate linkages mayalso be used, with these linkages being less degradable than succinate,glutarate or adipate linkages. Branched, cyclic or other hydrophobiclinkages may also be used. Polyethylene glycols and other precursors maybe prepared with these groups. The crosslinked hydrogel degradation mayproceed by the water-driven hydrolysis of the biodegradable segment whenwater-degradable materials are used. Polymers that include esterlinkages may also be included to provide a desired degradation rate,with groups being added or subtracted near the esters to increase ordecrease the rate of degradation. Thus it is possible to construct ahydrogel with a desired degradation profile, from a few days to manymonths, using a degradable segment. If polyglycolate is used as thebiodegradable segment, for instance, a crosslinked polymer could be madeto degrade in about 1 to about 30 days depending on the crosslinkingdensity of the network. Similarly, a polycaprolactone based crosslinkednetwork can be made to degrade in about 1 to about 8 months. Thedegradation time generally varies according to the type of degradablesegment used, in the following order:polyglycolate<polylactate<polytrimethylene carbonate<polycaprolactone.Thus it is possible to construct a hydrogel with a desired degradationprofile, from a few days to many months, using a degradable segment.

A biodegradable linkage in the organogel and/or xerogel and/or hydrogeland/or precursor may be water-degradable or enzymatically degradable.Illustrative water-degradable biodegradable linkages include polymers,copolymers and oligomers of glycolide, dl-lactide, 1-lactide, dioxanone,esters, carbonates, and trimethylene carbonate. Illustrativeenzymatically biodegradable linkages include peptidic linkages cleavableby metalloproteinases and collagenases. Examples of biodegradablelinkages include polymers and copolymers of poly(hydroxy acid)s,poly(orthocarbonate)s, poly(anhydride)s, poly(lactone)s,poly(aminoacid)s, poly(carbonate)s, and poly(phosphonate)s.

If it is desired that a biocompatible crosslinked matrix bebiodegradable or absorbable, one or more precursors having biodegradablelinkages present in between the functional groups may be used. Thebiodegradable linkage optionally also may serve as the water solublecore of one or more of the precursors used to make the matrix. For eachapproach, biodegradable linkages may be chosen such that the resultingbiodegradable biocompatible crosslinked polymer will degrade or beabsorbed in a desired period of time.

Matrix materials may be chosen so that degradation products are absorbedinto the circulatory system and essentially cleared from the body viarenal filtration. The matrix materials may be hydrogels in aphysiological solution. One method is to choose precursors that are notbroken down in the body, with linkages between the precursors beingdegraded to return the precursors or precursors with small changescaused by the covalent crosslinking process. This approach is incontrast to choosing biological matrix materials that are destroyed byenzymatic processes and/or materials cleared by macrophages, or thatresult in by-products that are effectively not water soluble. Materialsthat are cleared from the body by renal filtration can be labeled anddetected in the urine using techniques known to artisans. While theremight be at least a theoretical loss of some of these materials to otherbodily systems, the normal fate of the material is a kidney clearanceprocess. The term essentially cleared thus refers to materials that arenormally cleared through the kidneys.

Administration

Administration of a xerogel may be performed directly into the site ofinterest. For example, a lenticule of xerogel may be applied to acornea, or a film may be applied to a dermis or epidermis. Xerogelparticles may be administered by inhalation. And powder-delivery systemsmay be used to directly inject xerogel powders into a tissue.

Administration of a xerogel may also involve hydration at about the timeof use, or at the point of use. The xerogel is exposed to an aqueoussolution, for instance a physiological saline, and allowed to imbibewater to form a hydrogel. The hydrogel is implanted, either directly,surgically, or by injection through a syringe or catheter.

Embodiments of the invention include administration at or near an eye.The structure of the mammalian eye can be divided into three main layersor tunics: the fibrous tunic, the vascular tunic, and the nervous tunic.The fibrous tunic, also known as the tunica fibrosa oculi, is the outerlayer of the eyeball consisting of the cornea and sclera. The sclera isthe supporting wall of the eye and gives the eye most of its whitecolor. It is extends from the cornea (the clear front section of theeye) to the optic nerve at the back of the eye. The sclera is a fibrous,elastic and protective tissue, composed of tightly packed collagenfibrils, containing about 70% water.

Overlaying the fibrous tunic is the conjunctiva. The conjunctiva is amembrane that covers the sclera (white part of the eye) and lines theinside of the eyelids. It helps lubricate the eye by producing mucus andtears, although a smaller volume of tears than the lacrimal gland. Theconjunctiva is typically divided into three parts: (a) Palpebral ortarsal conjunctivam which is the conjunctiva lining the eyelids; thepalpebral conjunctiva is reflected at the superior formix and theinferior formix to become the bulbar conjunctiva, (b) Formixconjunctiva: the conjunctiva where the inner part of the eyelids and theeyeball meet, (c) Bulbar or ocular conjunctiva: the conjunctiva coveringthe eyeball, over the sclera. This region of the conjunctiva is boundtightly and moves with the eyeball's movements. The conjunctivaeffectively surrounds, covers, and adheres to the sclera. It is hascellular and connective tissue, is somewhat elastic, and can be removed,teased away, or otherwise taken down to expose a surface area of thesclera.

The vascular tunic, also known as the tunica vasculosa oculi, is themiddle vascularized layer which includes the iris, ciliary body, andchoroid. The choroid contains blood vessels that supply the retinalcells with oxygen and remove the waste products of respiration. Thenervous tunic, also known as the tunica nervosa oculi, is the innersensory which includes the retina. The retina contains thephotosensitive rod and cone cells and associated neurons. The retina isa relatively smooth (but curved) layer. It does have two points at whichit is different; the fovea and optic disc. The fovea is a dip in theretina directly opposite the lens, which is densely packed with conecells. The fovea is part of the macula. The fovea is largely responsiblefor color vision in humans, and enables high acuity, which is necessaryin reading. The optic disc is a point on the retina where the opticnerve pierces the retina to connect to the nerve cells on its inside.

The mammalian eye can also be divided into two main segments: theanterior segment and the posterior segment. The anterior segmentconsists of an anterior and posterior chamber. The anterior chamber islocated in front of the iris and posterior to the corneal endotheliumand includes the pupil, iris, ciliary body and aqueous fluid. Theposterior chamber is located posterior to the iris and anterior to thevitreous face where the crystalline lens and zonules fibers arepositioned between an anterior and posterior capsule in an aqueousenvironment.

The cornea and lens help to converge light rays to focus onto theretina. The lens, behind the iris, is a convex, springy disk whichfocuses light, through the second humour, onto the retina. It isattached to the ciliary body via a ring of suspensory ligaments known asthe Zonule of Zinn. The ciliary muscle is relaxed to focus on an objectfar away, which stretches the fibers connecting it with the lens, thusflattening the lens. Light enters the eye, passes through the cornea,and into the first of two humors, the aqueous humour. Approximatelytwo-thirds of the eye's total refractive power comes from the corneawhich has a fixed curvature. The aqueous humor is a clear mass whichconnects the cornea with the lens of the eye, helps maintain the convexshape of the cornea (necessary to the convergence of light at the lens)and provides the corneal endothelium with nutrients.

The posterior segment is located posterior to the crystalline lens andin front of the retina. It represents approximately two-thirds of theeye that includes the anterior hyaloid membrane and all structuresbehind it: the vitreous humor, retina, c, and optic nerve. On the otherside of the lens is the second humour, the vitreous humour, which isbounded on all sides: by the lens, ciliary body, suspensory ligamentsand by the retina. It lets light through without refraction, helpsmaintain the shape of the eye and suspends the delicate lens.

FIG. 8 shows certain points of delivery at or near eye 200. Eye 200includes sclera 212, iris 214, cornea 222, vitreous body 232, zonularspaces 242, fovea 236, retina 238, and optic nerve 225. One area fordelivery is topically at 260, with area 260 being indicated by dots onsurface of eye 200. Another area is intravitreally as indicated bynumeral 262, or trans-sclerally, as indicated by numeral 264. In use,for example a syringe 266, catheter (not shown) or other device is usedto deliver a xerogel (or gel or hydrogel or a precursors thereof),optionally through needle 268, into the eye, either intravitrealy, as at262 or peri-ocularly, as at 272. Another area is subconjunctivally (notshown), below the conjunctiva 211 and above the sclera 212. Drugs orother therapeutic agents are released to the intra-ocular space. In thecase of back-of-the-eye diseases, drugs may be targeted via theperi-ocular or intravitreal route to target approximate area 274, wherethey interact with biological features to achieve a therapy. Anembodiment is placement of a xerogel in contact with retina 238 or nearretina 238 without contacting it. For instance, xerogels, hydrogelsand/or particles (or rods, microspheres, a single material, beads, orother shapes set forth herein) may be delivered to a location adjacentto, or upon, retina 238. The hydrogel advantageously is anchored in thevitreous gel and does not allow diffusion of the particles. In contrast,other systems that use a rod or slippery microspheres do not provideanchoring and diffusion or migration in response to movement of, orrubbing of, the eye. The placement of the depot at or near the retina(or other location) allows a high concentration to be achieved at theintended site, with small particles being usable to deliver the drugsfor effective treatment. In contrast, spheres, rods, or other shapesthat are too large to diffuse or migrate have a volume/surface arearation that is unfavorable for effective controlled release. Anotherarea for placement of a xerogel, hydrogel and/or particles, or othermaterials comprising the particles is in a punctum (not shown), e.g., byplacing particles in a punctal plug (silicone, polysaccharide, hydrogel,or other material) that is inserted into a punctum of an eye.

Sites where drug delivery depots may be formed in or near an eye includethe anterior chamber, the vitreous (intravitreal placement), episcleral,in the posterior subtenon's space (inferior formix), subconjunctival, onthe surface of the cornea or the conjunctiva, among others. Perioculardrug delivery of an ophthalmic hydrogel implant using subconjunctival,retrobulbar or sub-Tenon's placement has the potential to offer a saferand enhanced drug delivery system to the retina compared to topical andsystemic routes.

An example of in situ placement is illustrated for an intravitrealimplant in FIG. 9A. In FIG. 9A, a xerorogel implant is injectedintravitrealy about 2.5 mm posterior to the limbus through a pars planaincision 390 using a sub-retinal cannula 392, as shown by depiction ofmagnifying glass 394 held so as to visualization incision 390 on eye310, which may be made following dissecting-away or otherwise clearingthe conjunctiva, as needed. A sub-retinal cannula 392 (or otherappropriate cannulas) is then inserted through incision 390 andpositioned intraocularly to the desired target site, e.g., at least oneof sites 396, 398, 300 (FIG. 9B) where the xerogel(s) are introduced andsubsequently form a hydrogel in situ. The xerogels form into anabsorbable gel 302, 304, and/or 306, adhering to the desired targetsite. Particles comprising a therapeutic agent may be included in thegel or gels. Significantly, it is possible to use a fine gauge needle toplace the precursors. Embodiments include placement with a 25 gaugeneedle. Further embodiments include using a needle smaller in diameterthan 25 gauge, e.g., 26, 27, 30, 31, 32 gauge.

Intravitreal in situ implant embodiments can improve the efficacy andpharmacokinetics of potent therapeutic agents in the treatment of eyediseases and minimize patient side effects in several ways. First, theimplant can be placed in the vitreous cavity at a specific disease site,bypassing the topical or systemic routes and thereby increasing drugbioavailability. Secondly, the implant maintains local therapeuticconcentrations at the specific target tissue site over an extendedperiod of time. Thirdly, the number of intravitreal injections would besubstantially reduced over a 12 month therapy regimen, thereby reducingpatient risk of infection, retinal detachment and transient visualacuity disturbances (white specks floating in the vitreous) that canoccur until the drug in the vitreous migrates down toward the inferiorwall of the eye and away from the portion of the central vitreous ormacula.

The xerogels or the xerogels-hydrated-as-hydrogels (thexerogel/hydrogels) may be placed on scleral tissue either with orwithout the presence of the conjunctiva. The xerogel/hydrogels may beadhered to the sclera or other tissue near the sclera to promote drugdiffusion through the intended tissue or to provide a stable depot todirect the therapeutic agents as required. A hydrogel adhesive such asRESURE® sealant may be employed as an adhesion aid. In some embodiments,the conjunctiva of the eye may be removed, macerated, dissected away, orteased-free so that the tissue can be lifted away from the sclera toaccess a specific region of the sclera for implantation or injection ofthe xerogel/hydrogels. A xerogel/hydrogel is placed to make a layer on,and adhere to, the surface. The conjunctiva may be allowed to contactthe tissue if it is still present or retains adequate mechanicalintegrity to do so. In some embodiments the xerogel/hydrogels iscomprised of at least 50%, 75%, 80%, 90%, or 99% w/w water-solubleprecursors (calculated by measuring the weight of the hydrophilicprecursors and dividing by the weight of all precursors, so that theweight of water or solvents or non-hydrogel components is ignored) toenhance the non-adhesive properties of the hydrogel. In someembodiments, such hydrophilic precursors substantially comprise PEOs. Insome embodiments, drugs to reduce tissue adherence mediated bybiological mechanisms including cell mitosis, cell migration, ormacrophage migration or activation, are included, e.g.,anti-inflammatories, anti-mitotics, antibiotics, PACLITAXEL, MITOMYCIN,or taxols.

In other embodiments, the sclera is not substantially cleared of theconjunctiva. The conjunctiva is a significant tissue mass that overlaysmuch or all of the sclera. The conjunctiva may be punctured orpenetrated with a needle or catheter or trocar and precursors introducedinto a space between the sclera and conjunctiva. This placement of theimplant is referred to as a subconjunctival location. In some cases theconjunctiva may be punctured to access a natural potential space betweenthe tissues that is filled by the precursors. In other cases, apotential or actual space is created mechanically with a trocar,spreader, or the like, that breaks the adherence between the sclera andconjunctiva so that precursors may be introduced. The conjunctiva hasenough elasticity to allow useful amounts of a xerogel to be introducedor forced into such natural or created spaces. Accordingly, in somecases, the xerogel/hydrogel volume is between about 0.25 to about 5 ml;artisans will immediately appreciate that all the ranges and valueswithin the explicitly stated ranges are contemplated, e.g., about 1 mlor from 0.5 ml to about 1.5 nil.

Moreover, removal of a xerogel that has formed a hydrogel, whetherpresent intraocularly or periocularly, is also readily achieved usingeither a vitrectomy cutter if the implant is located in the vitreouscavity or a manual I/A syringe and cannula if the implant is located onthe scleral surface or irrigation/aspiration handpiece. This contrastswith major surgical procedures needed for the removal of someconventional non-absorbable implants.

In further embodiments, a xerogel/hydrogel material may be placed intothe patient, e.g., in a tissue or organ, including subcutaneous,intramuscular, intraperitoneally, in a potential space of a body, or ina natural cavity or opening. The material provides a depot for releaseof an agent over time. Embodiments thus include between about 0.5 andabout 500 ml volumes for placement (referring to total volume in thecase of particle collections delivered); artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated, e.g., from 1 to 10 ml or from 5 to 50 ml.Intraperitoneal or intramuscular injection, for instance, is a usefularea for extended control release of agents over hours, days, or weeks.

The materials described herein may be used to deliver drugs or othertherapeutic agents (e.g., imaging agents or markers). One mode ofapplication is to apply a mixture of xerogel/hydrogel particles andother materials (e.g., therapeutic agent, buffer, accelerator,initiator) through a needle, cannula, catheter, or hollow wire to asite. The mixture may be delivered, for instance, using a manuallycontrolled syringe or mechanically controlled syringe, e.g., a syringepump. Alternatively, a dual syringe or multiple-barreled syringe ormulti-lumen system may be used to mix the xerogel/hydrogel particles ator near the site with a hydrating fluid and/or other agents.

The xerogels may be provided in flowable form to the site, e.g., asflowable particles. The xerogels may be suspended in a liquid andapplied to the site. The xerogel particles may be made to have a maximumdiameter for manual passage out of a syringe through a 3 to 5 Frenchcatheter, or a 10 to 30 gauge needle. Artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated, e.g., 25 to 30 gauge. The use of small needlesis particular advantageous in the eye, which is a sensitive organ.Applications to other organs are also advantageous, e.g., to controlbleeding or other damage. The particles may be formed by creating ahydrogel and then breaking it up into smaller pieces. The material maybe, e.g., ground in a ball mill or with a mortar and pestle, or choppedor diced with knives or wires. Or the material may be cut-up in ablender. Another process involves forcing the material in the organogelor gel step through a mesh, collecting the fragments, and passing themthrough the same mesh or another mesh until a desired size is reached,followed by making the xerogel. The xerogel/hydrogel may contain thetherapeutic agent-loaded particles. Some or all of the hydrogelparticles may contain the therapeutic agent-loaded particles. In someembodiments, a first set of therapeutic agent-loaded particles loadedwith a first therapeutic agent is included inside a first set of xerogelparticles and a second set of therapeutic agent-loaded particles loadedwith a second therapeutic agent is included inside a second set ofxerogel particles. In this manner, a plurality of agents may be releasedfrom a single implant. Embodiments of the particles include those with aparticular shape such as sphere, rod, or disc.

Embodiments include placement of a plurality of xerogel/hydrogelparticles. The xerogel/hydrogel particles may comprise a therapeuticagent, e.g., a protein such as an anti-VEGF. The particles may be madewith a sized for manual passage through a 27-gauge or smaller diameterneedle. The pressure to force the particles through the needle may beprovided manually.

An alternative to delivery of particles is to pre-form the gel as ashaped article and then introduce the material into the body. Forexample, the xerogel/hydrogels may be formed as spheres, rods,cylinders, or other shapes. Embodiments include solid rods ofxerogel/hydrogels for subcutaneous implantation and delivery of one ormore agents.

Xerogel/hydrogels as set forth herein may be used for tissueaugmentation. The use of collagen as for dermal augmentation is wellknown. Xerogel/hydrogels, for example particulates, may be used fordermal filler or for tissue augmentation. Embodiments include injectingor otherwise placing a plurality of particles in a tissue, or forming ahydrogel in situ. The material may be injected or otherwise placed atthe intended site.

Xerogel/hydrogels as set forth herein may be used to separate tissues toreduce a dose of radioactivity received by one of the tissues. As setforth in U.S. Pat. No. 7,744,913, which is hereby incorporated byreference herein for all purposes with the present specificationcontrolling in case of conflict, spacer materials may be placed in apatient. Certain embodiments are a method comprising introducing aspacer to a position between a first tissue location and a second tissuelocation to increase a distance between the first tissue location andthe second tissue location. Further, there may be a step ofadministering a dose of radioactivity to at least the first tissuelocation or the second tissue location. A method, for example, isdelivering a therapeutic dose of radiation to a patient comprisingintroducing a biocompatible, biodegradable particulate xerogel, e.g., acollection of particles optionally with radioopaque contents, between afirst tissue location and a second tissue location to increase adistance between the first tissue location and the second tissuelocation, and treating the second tissue location with the therapeuticdose of radiation so that the presence of the filler device causes thefirst tissue location to receive less of the dose of radioactivitycompared to the amount of the dose of radioactivity the first tissuelocation would receive in the absence of the spacer. The spacer may beintroduced as a xerogel that forms a hydrogel in the patient that isremoved by biodegradation of the spacer-hydrogel in the patient. Anexample is the case wherein the first tissue location is associated withthe rectum and the second tissue location is associated with theprostate gland. The amount of reduction in radiation can vary.Embodiments include at least about 10% to about 90%; artisans willimmediately appreciate that all the ranges and values within theexplicitly stated ranges are contemplated, e.g., at least about 50%. Theradiation may alternatively be directed to a third tissue so that thefirst tissue or the second tissue received a lower amount of radiationas a result of its separation from the other tissue(s). The first tissueand the second tissue may be adjacent to each other in the body, or maybe separate from each other by other tissues. Spacer volumes forseparating tissues are dependent on the configuration of the tissues tobe treated and the tissues to be separated from each other. In manycases, a volume of about 20 cubic centimeters (cc's or mls) is suitable.In other embodiments, as little as about 1 cc might be needed. Othervolumes are in the range of about 5-1000 cc; artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated, e.g., 10-30 cc. In some embodiments, spacersare administered in two doses at different times so as to allow thetissues to stretch and accommodate the spacer and thereby receive alarger volumes of spacer than would otherwise be readily possible.Tissues to be separated by a spacer include, for example, at least oneof a rectum, prostate, and breast, or a portion thereof. For instance, afirst portion of a breast may be separated from a second portion.

Kits

Kits or systems for making hydrogels from a xerogel may be prepared sothat the xerogels are stored in the kit and made into a hydrogel whenneeded for use with a patient. And kits may be made for applying axerogel in a xerogel form. Applicators may be used in combination withthe xerogel and/or hydrogel. The kits are manufactured using medicallyacceptable conditions and contain components that have sterility, purityand preparation that is pharmaceutically acceptable. The kit may containan applicator as appropriate, as well as instructions. Xerogel particlescomprising a therapeutic agent may be available for mixing with asolution that is in the kit or provided separately. The xerogelcomponents may be provided as: one or more containers of a xerogel thatform a hydrogel, with the xerogel being in the form of a plurality ofparticles that are placed into the patient, or as a unitary implant.Solvents/solutions may be provided in the kit or separately, or thecomponents may be pre-mixed with the solvent. The kit may includesyringes and/or needles for mixing and/or delivery. The kit or systemmay comprise components set forth herein.

Some embodiments provide a single applicator, e.g., one syringe, thatcomprises xerogel particles for delivery, with an aqueous solution beingadded to the applicator for hydration, followed by use of the syringe toplace the materials in a patient. The xerogel particle solvent may beessentially water, meaning about 99% v/v of the solvent is water, withsalts or buffers being present as desired. Other solvents may be usedthat are safe and biocompatible, e.g., dimethylsulfoxide. The xerogelparticles may further comprise powders of proteins and/or other agents.

Packaging for a precursor and/or for an entire kit is preferablyperformed under dry conditions that are oxygen-free. The precursorsand/or kit components may be placed in a hermetically sealed containerthat is not permeable to moisture or oxygen, for instance, glass ormetal (foil) containers.

The xerogels containing the protein powder, or other solid phase, watersoluble biologics, may be gamma sterilized at the end of the implantablematerial manufacturing process. Alternatively or furthermore there maybe a sterilization process either before and/or after assembly andsealing of a kit. Low moisture conditions are often helpful in thistechnique. It has been observed that the solid phase dispersed powdersresist the formation of aggregates and crosslinking under gammaradiation. This result is unexpected and surprising since gammaradiation sterilization is generally believed to harm protein or peptidebiologics. Without being bound to a particular theory of operation, itis believed that the small particle size and absence of moisturedisfavors these unwanted reactions.

Further Description

(1) A first embodiment of the invention is directed to a process ofmaking a medical material comprising forming an organogel around apowder of a water soluble biologic, with the powder being dispersed inthe organogel. (2) A second embodiment of the invention is directed to aprocess of making a medical material comprising forming an gel around apowder of a water soluble biologic, with the powder being dispersed inthe gel, wherein forming the gel comprises preparing a melt of one ormore precursors and covalently crosslinking the precursors. (3) A thirdembodiment of the invention is directed to process of making a medicalmaterial comprising forming an organogel around particles of a powder ofa biologic, with the particles being dispersed within the organogel, andremoving solvents from the organogel, thereby forming a xerogel, saidprocess being performed in an absence of water. (4) A fourth embodimentof the invention is directed to a process of making a medical materialcomprising forming an organogel or a gel from a melt, making a xerogelfrom the (organo)gel, and providing the xerogel as a collection ofparticles, wherein the xerogel is a hydrogel upon exposure to an aqueoussolution. (5) A fifth embodiment of the invention is directed to apharmaceutically acceptable material as in any of embodiments I-IV. (6)A sixth embodiment of the invention is directed to a medical materialcomprising a pharmaceutically acceptable biodegradable xerogelcomprising dispersed protein particles, the protein being a therapeuticagent and having a secondary and/or a tertiary structure. Further, saidprotein may be released from the particles in aqueous solution in aconformation that is substantially free of denaturation. (7) A seventhembodiment of the invention is directed to a (pharmaceuticallyacceptable) biomaterial for controlled release of a therapeutic watersoluble biologic comprising a pharmaceutically acceptable xerogel thatcomprises solid particles of the biologic dispersed therein,(optionally, with the xerogel being free of hydrophobic materials) andwith the xerogel being a hydrogel when exposed to water. (8) An eighthembodiment is a method of making any of the materials of embodiments VIor VII.

Further embodiments are: (9) as in any of 1-8 wherein the (watersoluble) biologic is a protein (10) as in any of 1-9 wherein the proteinhas a molecular mass of at least about 10,000 Daltons and a sugar isassociated with the protein (11) as in any of 1-10 wherein the powder isused and is a first powder, with the process further comprising a secondpowder that comprises a second water soluble biologic agent, with thefirst powder and the second powder being dispersed through the organogel(12) as in any of 1-11 wherein the powder is used and has an averageparticle size between about 10 μm and about 10 μm (13) as in any of 1-12wherein the organogel is formed in an absence of aqueous solution (14)as in any of 1-13 comprising removing solvents from the organogel as maybe needed to thereby form a xerogel (15) as in any of 1-14 comprisingremoving solvents by a process chosen from the group consisting ofvacuum removal, lyophilization, and freezing followed by application ofa vacuum (16) as in any of 1-15 comprising the xerogel, wherein thexerogel is a hydrogel upon exposure to an aqueous solution (17) as inany of 1-15 comprising the powder, wherein the (water soluble) biologicsremain substantially in the powder, in a solid phase, when the hydrogelis formed, and slowly dissolve over a period of time when the hydrogelis exposed to physiological solution in vivo in a mammal (18) as in 17with said dissolving being in a period of time is in a range from about1 week to about 52 weeks (19)) as in any of 1-18 wherein the biologic inthe gel is a protein having a secondary and/or a tertiary structure,with the protein being released in a conformation that is substantiallyfree of denaturation as measurable by, for example, enzyme-linkedimmunosorbent assay and isoelectric focusing (20) as in any of 1-19wherein the gel or organogel or xerogel comprises covalently crosslinkedhydrophilic polymers (21) as in any of 1-20 wherein the gel organogel orxerogel organogel comprises covalently crosslinked hydrophilic polymerschosen from the group consisting of polyethylene oxide, polyvinylpyrrolidinone, hyaluronic acid, polyhydroxyethylmethacrylate, and blockcopolymers thereof (22) as in any of 1-21 wherein, when the hydrogel ispresent, the hydrogel is biodegradable by spontaneous hydrolysis ofhydrolytically degradable linkages chosen from the group consisting ofesters, carbonates, anhydrides and orthocarbonates (23) as in any of1-22 wherein, when the organogel is present, the organogel comprisesblock copolymers that form the organogel and that, after the solventsare removed to form a xerogel, form a hydrogel upon exposure to anaqueous solution (24) as in any of 1-23 wherein, when the organogel ispresent, the organogel comprises wherein the organogel (and thehydrogel) comprises ionically crosslinked polymers (25) as in any of1-24 wherein, when the organogel is present, the organogel comprises amember chosen from the group consisting of alginate, gellan, collagen,and polysaccharide (25) as in any of 1-24 comprising forming a pluralityof particles out of: (a) the gel (b) the organogel (c) a xerogel madefrom the gel or the organogel, or (d) a hydrogel made from the gel ororganogel (26) as in any of 1-25 wherein, when the organogel is present,forming the organogel from a precursor in an organic solvent, with theprecursor being chemically reacted to form covalent bonds to therebyform the organogel, wherein the organogel is covalently crosslinked (27)as in any of 1-26 wherein the precursor is reacted by free radicalpolymerization to form the organogel (28) as in any of 1-27 wherein theprecursor is a first precursor comprising a first functional group andfurther comprising a second precursor comprising a second functionalgroup, with the first functional group and the second functional groupbeing reactive in the organic solvent to form the covalent bonds (29) asin 28 wherein the first functional group and the second functional groupare each chosen from the group consisting of electrophile andnucleophile, and the reaction between the first functional group andsecond functional group is an electrophilic-nucleophilic reaction thatforms the covalent bond (30) as in 28 or 29 wherein the electrophilicgroup comprises succimide, succinimide ester, n-hydroxysuccinimide,maleimide, succinate, nitrophenyl carbonate, aldehyde, vinylsulfone,azide, hydrazide, isocyanate, diisocyanate, tosyl, tresyl, orcarbonyldiimidazole (31) as in any of 28-30 wherein the nucleophilegroup comprises a primary amine or a primary thiol (32) as in any of28-31 wherein the first precursor and the second precursor are watersoluble (33) as in any of 28-32 wherein at least one of the firstprecursor and the second precursor comprises a synthetic polymer (34) asin any of 28-33 wherein the first precursor comprises a polymer chosenfrom the group consisting of polyethylene glycol, polyacrylic acid,polyvinylpyrrolidone, and block copolymers thereof (35) as in any of1-34 comprising the organogel, comprising preparing the organogel as astructure chosen from the group consisting of a rod, a sheet, aparticle, a sphere, and a collection of at least one of the same (36) asin any of 1-35 comprising, or further comprising a therapeutic agent,wherein the agent comprises a fluoroquinolone, moxifloxacin, travoprost,dexamethasone, an antibiotic, or a vestibulotoxin (37) as in 36 with theorganogel further comprising a permeation enhancer (38) as in any of 1-8wherein the organogel is physically crosslinked by formation of domains,the process further comprising forming the organogel from a precursor inan organic solvent, with the precursor being a block copolymer thatcomprises a first block and a second block (39) as in 38 comprisingheating a mixture of the precursor and the organic solvent and allowingthe solution to cool, thereby precipitating at least the first block ofthe copolymeric precursor, with said domains comprising at least thefirst block (40) as in 38 or 39 comprising mixing the precursor in afirst organic solvent that dissolves the copolymeric precursor, with allof the blocks of the copolymeric precursor being soluble in the firstorganic solvent, and adding a second organic solvent that is misciblewith the first organic solvent, with the first block of the copolymericprecursor being insoluble in the second organic solvent, with the secondsolvent being effective to form the domains, with the domains comprisingthe first block of the copolymer (41) as in any of 38-40 wherein thecopolymeric precursor comprises a block chosen from the group consistingof polyethylene glycol (42) as in any of 38-41 wherein the copolymericprecursor further comprises a second block chosen from the groupconsisting of polylactic acid, polyglycolic acid, polytrimethylenecarbonate, polydioxanone, polyalkyl, polybutylene terephthalate, andpolylysine (43) as in any of 1-37 wherein the organogel is free ofhydrophobic materials; alternatively being free of hydrophobic polymers,or being free of all hydrophobic materials with the exception ofsolvents (which may be somewhat hydrophobic) (44) as in any of 1-43comprising preparing a powder of the biologic according to a method thatavoids denaturation of the biologic, and, once the powder has beenprepared, preventing exposure of the powder to water (45) as in any of1-44 wherein the biologic is therapeutic protein having a secondaryand/or tertiary structure (46) as in any of 1-45 comprising a xerogel,wherein the xerogel is a hydrogel after being exposed to water (47) asin any of 1-46 wherein the hydrogel, or a hydrogel made from thegel/organogel/xerogel is biodegradable (48) as in any of 1-47 comprisingthe xerogel, wherein a cumulative amount of release of the agent reaches90% w/w of the agent at a time between about 1 month and about 6 monthsafter placement of the hydrogel and particles in a saline solution (49)a biomaterial as in any of 1-48 (50) a biomaterial as in any of 1-49wherein the xerogel comprises covalently crosslinked hydrophilicpolymers (51) a biomaterial as in any of 1-50 wherein the water solublebiologic is a protein having a secondary and/or tertiary structure (52)a biomaterial as in any of 1-51 wherein the water soluble biologicremains substantially in the solid phase, when the hydrogel is formed,and slowly dissolves over a period of time when the hydrogel is exposedto physiological solution in vivo in a mammal (53) a biomaterial as inany of 1-52 comprising the organogel, wherein the organogel comprisescovalently crosslinked hydrophilic polymers (54) the biomaterial of 53wherein the polymers comprise a member chosen from the group consistingof polyethylene oxide, polyvinyl pyrrolidinone, hyaluronic acid,polyhydroxyethylmethacrylate, and block copolymers thereof (54) as inany of 1-53 with the material being a structure chosen from the groupconsisting of a rod, a sheet, a particle, a sphere, and a collectionthereof (55) any of 1-54 comprising the xerogel, or a process ofproviding the xerogel as, a collection of particles, e.g., by a methodchosen from the group consisting of (a) making the organogel andbreaking it up to form particles for the collection, (b) making thexerogel and breaking up the xerogel to form particles for thecollection, and (c) making the organogel as a plurality of particles forthe collection, said particles being stripped of the organic solvent(s)to make the xerogel (56) a process as in 55 comprising making aplurality of the collections of particles, with the collections havingdifferent rates of degradation in vivo, and mixing collections to make abiomaterial having a degradation performance as desired.

These embodiments 1-56 may further be prepared as a kit with thepolymers, biologic or protein, and an applicator, with the kit being ina sterile container. These embodiments 1-56 may be further practiced byplacing the material, or a material made by one of the processes, incontact with a tissue of a patient. Examples of the tissues are anintraperitoneal space, a muscle, a dermis, an epidermis, a natural lumenor void, an abdominal cavity, a prostate, a rectum, a location between aprostate and a rectum, a breast, a tissue between a radiation target andhealthy tissue, and a vasculature.

EXAMPLES Example 1 Preparation of Organogels and Xerogels ContainingProtein Particles

Polyethylene Glycol (PEG) Compounds

PEG compounds were obtained with the following structures:

TABLE 1 PEG Esters PEG Molecular Number weight of PEG End group (Da)arms moiety Reactive end group Designation 15000 8 Succinic acidN-hydroxysuccinimide 8a15KSS 20000 4 Glutaric acid N-hydroxysuccinimide4a20KSG 15000 8 Glutaric acid N-hydroxysuccinimide 8a15KSG 20000 4Adipic acid N-hydroxysuccinimide 4a20KSAP 20000 4 GlutaricN-hydroxysuccinimide 4a20KSGA amide 20000 8 None Free amine 8a20KA or8a20KNH2Preparation of PEG Solutions

PEG powders were weighed out and put in a 10 ml graduated cylinder as inthe following Tables:

TABLE 2 Preparation of PEG Ester solutions in Methylene Chloride ExamplePEG Ester (g) 1A-1 8a15kSS 0.86 1B-1 4a20kSG 1.33 1C-1 8a15kSG 0.86 1D-14a20kSAP 1.33 1E-1 4a20kSGA 1.33

TABLE 3 Preparation of PEG Amine solutions in Methylene Chloride ExamplePEG Amine (g) 1A-2 8a20KNH2 1.14 1B-2 8a20KNH2 0.67 1C-2 8a20KNH2 1.141D-2 8a20KNH2 0.67 1E-2 8a20KNH2 0.67

Methylene chloride was added to the 10 mL mark once the PEG wasdissolved.

Preparation of Ground Ovalbumin

In a nitrogen-filled glove bag, ovalbumin (Worthington BiochemicalCorporation; LS003048) was ground using a mortar and pestle and sievedto less than 20 μm particles through a stainless steel sieve.

Preparation of Ovalbumin Organogels

Ground ovalbumin was weighed in a polyethylene female LUER-LOK syringe.PEG Amine solution was mixed with the ovalbumin to form a suspension.PEG Ester solution was put in a male polyethylene luer Lock syringe. Thesyringes were mated and solutions were mixed syringe-to-syringe for 10seconds and allowed to stand in the male syringe for 10 minutes, duringwhich time was formed a gel containing the protein. The syringe was cutopen and the gel-protein cylinder was removed. The gels were place undervacuum overnight to dry. The following Table summarizes the samplesprepared in this manner.

TABLE 4 Albumin Organogel Preparation PEG Ester PEG Amine Protein AmountAmount ovalbumin Example Example (μL) Example (μL) (mg) 1A-3 1A-1 5001A-2 500 103.5 1B-3 1B-1 500 1B-2 500 106 1C-3 1C-1 500 1C-2 500 105.11D-3 1D-1 500 1D-2 500 102.4 1E-3 1E-1 500 1E-2 500 100.2Preparation of Ovalbumin-PEG Xerogels

The syringe containing the ovalbumin organogel was cut open and thegel-protein cylinder was removed. The gels were placed under vacuumovernight to dry. Dried xerogels were stored under nitrogen headspace at5° C.

Preparation of Ground Rabbit IgG

In a nitrogen-filled glove bag, rabbit IgG (IgG from Rabbit serum;Sigma; >95%) was hand ground using a mortar and pestle and sieved toless than 20 μm through a stainless steel sieve.

Preparation of Rabbit IgG Organogels

Ground rabbit IgG was weighed in a polyethylene female luer locksyringe. PEG Amine solution was mixed with the ovalbumin to formsuspension. PEG Ester solution was put in male polyethylene LUER-LOKsyringe. The syringes were mated and solutions were mixedsyringe-to-syringe for 10 seconds and allowed to stand in the malesyringe for 10 minutes to form the gel containing protein. The syringewas cut open and the gel-protein cylinder was removed. The gels wereplace under vacuum overnight to dry. The table below summarizes thesamples prepared in this manner. The following Table summarizes thesamples prepared in this manner.

TABLE 5 Rabbit IgG Organogel preparation PEG Ester PEG Amine ProteinAmount Amount Rabbit IgG Example Example (μL) Example (μL) (mg) 1A-41A-1 100 1A-2 100 9.47 1B-4 1B-1 100 1B-2 100 9.52 1C-4 1C-1 100 1C-2100 9.78 1D-4 1D-1 100 1D-2 100 10.29 1E-4 1E-1 100 1E-2 100 10.4Preparation of Rabbit IgG-PEG Xerogels

The syringe containing the rabbit IgG organogel was cut open and thegel-protein cylinder was removed. The gels were place under vacuumovernight to dry. Dried xerogels were stored under nitrogen headspace at5° C.

Example 2 In Vitro Release of Proteins from Hydrogels

Stability of Protein in Buffer Solutions

Ovalbumin (Worthington Biochemical Corporation; LS003048) and rabbit IgG(IgG from Rabbit serum; Sigma; >95%) were dissolved in TRIS Buffer at0.065 mg/ml. Initial samples were taken for baseline and at various timepoints to determine protein stability in the buffer. Samples wereanalyzed for protein content by HPLC and ELISA. The results aresummarized in the tables below.

TABLE 6 HPLC Protein Stability Study (50 mL Tris Buffer, pH 8.5, shakingat 50 rpm) Elapsed Time Ovalbumin IgG (hr) recovered recovered 0.00100.0% 100.0% 2.00 98.3% 97.1% 6.00 97.2% 99.5% 24.00 95.5% 97.7% 48.0095.0% 98.3% 96.00 94.1% 93.3%

TABLE 7 ELISA Protein Stability Study (50 mL Tris Buffer, pH 8.5,shaking at 50 rpm, 37 C.) Elapsed Time Ovalbumin IgG (hr) recoveredrecovered  5 min 97.7% 109.5%  2 hour 99.5% 87.1%  6 hour 98.4% 85.5% 24hour 91.3% 76.0% 48 hour 99.9% 78.8% 96 hour 70.1% 83.8%

The results show the proteins are sufficiently stable for use withaccelerated in vitro protein release testing.

In Vitro Protein Sustained Release Study

Samples of xerogels from Example 1 were cut, weighed and added to 50 mlTRIS buffer in a 50 mL centrifuge tube. Stainless steel dissolutioncages were used to hold the sample in the bottom half of the centrifugetube. The tubes were submerged in a shaking water bath at 37° C. and 50RPM.

TABLE 8 Accelerated and Real-Time In Vitro Protein Release Study XerogelBuffer from protein in Buffer Temperature Example Example Protein sample(mg) pH (° C.) 2A 1A-3 ovalbumin 24.46 8.5 37 2B 1B-3 ovalbumin 23.778.5 37 2C 1C-3 ovalbumin 23.37 8.5 37 2D 1D-3 ovalbumin 22.82 8.5 37 2E1E-3 ovalbumin 20.22 8.5 37 2F 1A-3 ovalbumin 25.12 7.4 37 2G 1A-4 IgG5.11 8.5 37 2H 1B-4 IgG 8.84 8.5 37 2I 1C-4 IgG 9.95 8.5 37 2J 1D-4 IgG10.8 8.5 37 2K 1E-4 IgG 10.66 8.5 37

Buffer medium samples were taken at 2 hrs, 4 hrs, 8 hours and then every8 hours after that until the gel degraded. Buffer medium was fullyexchanged at every time point. The samples collected were analyzed byHPLC and ELISA. The results are shown graphically below in FIGS. 2-5.

Drug Release Profile Customization

Combinations of the various vehicles may be used to customize a releaserate for a therapeutic agent. The release rates for various particleswere combined and a composite total release rate was calculated, asdepicted in FIGS. 6 and 7. FIG. 6 depicts a substantially zero-orderrelease kinetics from about 10 to about 60 hours. FIG. 7 depicts afinely tuned system. There is a first release that provides an initialburst for the first 24 hours, followed by additional zero order releasefrom about 24 to about 100 hours. The zero-order release is sustainedthrough the final dissolution of the materials.

Example 3 Formation of a Crosslinked Gel from a Melt of Precursors

0.86 g of an 8-armed branched PEG of about 15,000 Daltons terminatedwith SS (8a15KSS) was melted at 50° C. 1.14 g of an 8-armed branched PEGof about 20,000 Daltons terminated with primary amines (8a20KNH2) wasweighed with 0.5 g of bovine serum albumin (BSA) powder in a 10 mlsyringe and then soaked in a water bath at 60° C. to melt 8a20KNH2. Adrop of the 8a15KSS melt was placed on a 50° C. hot plate surface nextto a drop of 8a20KNH2melt/BSA. Drops were mixed quickly to gel withinless than 2 seconds. Gels formed contain BSA particles in the solid formas observed by microscopy.

Formed gels were transferred to scintillation vials filled withTris-buffered physiological saline (TBS) pH8.5 buffer to rapidlyhydrolyze the polymer and release the BSA.

After gel degradation, the resulting TBS release media was noted to beclear indicating the solubility of BSA in TBS and did not showprocessing effects on the protein solubility in terms of aggregation ordenaturation.

Patents, patent applications, patent publications, and references setforth herein are hereby incorporated herein by reference for allpurposes; in the case of conflict, the instant specification controls.

The invention claimed is:
 1. A process of making a medical materialcomprising crosslinking a precursor to form an organogel around a powderof a water soluble biologic, with the organogel comprising, and theprecursor being crosslinked in, an organic solvent selected fromanhydrous and hydrophobic solvents, methylene chloride, or dimethylcarbonate, with the water soluble biologic being directly exposed to theorganic solvent and the powder being dispersed in the organogel.
 2. Theprocess of claim 1 wherein the water soluble biologic is a protein thathas a molecular mass of at least about 10,000 Daltons and a sugar isassociated with the protein.
 3. The process of claim 1 furthercomprising a second powder of a second water soluble biologic, with thepowder and the second powder being dispersed in the organogel.
 4. Theprocess of claim 1 wherein the organogel is formed in an absence ofaqueous solution.
 5. The process of claim 1 further comprising removingthe organic solvent from the organogel to thereby form a xerogel.
 6. Theprocess of claim 5 wherein the organic solvent is removed by a processchosen from the group consisting of vacuum removal, lyophilization, andfreezing followed by application of a vacuum.
 7. The process of claim 5wherein the xerogel is a hydrogel upon exposure to an aqueous solution.8. The process of claim 1, wherein the organogel comprises covalentlycrosslinked hydrophilic polymers.
 9. The process of claim 1 wherein theorganogel comprises ionically crosslinked polymers.
 10. The process ofclaim 1 wherein the organogel comprises a member chosen from the groupconsisting of alginate, gellan, collagen, and polysaccharide.
 11. Theprocess of claim 1, wherein the precursor is covalently crosslinked toform the organogel.
 12. The process of claim 11 wherein the precursor isreacted by free radical polymerization to form the organogel.
 13. Theprocess of claim 11 wherein the precursor is a first precursorcomprising a first functional group and a second precursor comprising asecond functional group, with the first functional group and the secondfunctional group being reactive in the organic solvent to form thecovalent crosslinks.
 14. The process of claim 13 wherein the firstfunctional group and the second functional group are each chosen fromthe group consisting of electrophile and nucleophile, and the reactionbetween the first functional group and second functional group is anelectrophilic-nucleophilic reaction that forms the covalent crosslinks.15. The process of claim 14 wherein the electrophilic group comprisessuccinimide, succinimide ester, n-hydroxysuccinimide, maleimide,succinate, nitrophenyl carbonate, aldehyde, vinylsulfone, azide,hydrazide, isocyanate, diisocyanate, tosyl, tresyl, orcarbonyldiimidazole.
 16. The process of claim 15 wherein the nucleophilegroup comprises a primary amine or a primary thiol.
 17. The process ofclaim 13 wherein the first precursor and the second precursor are watersoluble.
 18. The process of claim 13 wherein at least one of the firstprecursor and the second precursor comprises a synthetic polymer. 19.The process of claim 18 wherein the first precursor comprises a polymerchosen from the group consisting of polyethylene glycol, polyacrylicacid, polyvinylpyrrolidone, and block copolymers thereof.
 20. Theprocess of claim 1 wherein the precursor is physically crosslinked toform the organogel.
 21. The process of claim 1 comprising preparing thepowder of the water soluble biologic according to a method that avoidsdenaturation of the biologic, and, once the powder has been prepared,preventing exposure of the powder to water until the medical material isused with a patient.
 22. The process of claim 1 wherein the organicsolvent is an anhydrous and hydrophobic solvent.
 23. The process ofclaim 1 wherein the organic solvent is methylene chloride.
 24. Theprocess of claim 1 wherein the organic solvent is dimethyl carbonate.25. The process of claim 1 wherein an amount of the water solublebiologic in the powder is at least 50% w/w.
 26. The process of claim 1wherein an amount of the water soluble biologic in the powder is atleast 80% w/w.
 27. The process of claim 1 wherein the powder consistsessentially of the water soluble biologic.
 28. The process of claim 1wherein the water soluble biologic comprises a member of the groupconsisting of an antibody, an antibody fragment, and a short chainvariable fragment antibody.
 29. The process of claim 1 wherein the watersoluble biologic comprises a protein or a fusion protein.
 30. Theprocess of claim 1 wherein the water soluble biologic comprises a memberof the group consisting of a carbohydrate, a polysaccharide, a nucleicacid chain, an antisense nucleic acid, a ribonucleic acid (RNA), adeoxyribonucleic acid, a small interfering RNA, and an aptamer.
 31. Theprocess of claim 1 wherein the water soluble biologic comprises ananti-vascular endothelial growth factor (VEGF) agent.
 32. The process ofclaim 1 wherein the water soluble biologic provides for treatment ofmacular degeneration.
 33. The process of claim 1 wherein the watersoluble biologic comprises an anti-cancer agent.
 34. The process ofclaim 1 wherein the water soluble biologic comprises insulin.
 35. Theprocess of claim 1 wherein the water soluble biologic comprises a growthfactor.
 36. The process of claim 1 wherein the water soluble biologiccomprises a member of the group consisting of antibiotics,antineoplastic agents, hormones, angiogenic agents, anti-angiogenicagents, neurotransmitters, psychoactive drugs, chemotherapeutic drugs,drugs affecting reproductive organs, and genes.
 37. The process of claim1 wherein the water soluble biologic is for treatment of a conditionselected from the group consisting of elevated intraocular pressure,fungal blepharitis, conjunctivitis, keratitis, open-angle glaucoma,ocular hypertension, inflammation of an eye, keratoconjunctivitis, andallergic conjunctivitis.
 38. A process of making a medical materialcomprising crosslinking a precursor to form an organogel around a powderof a water soluble biologic, with the organogel comprising, and theprecursor being crosslinked in, an organic solvent selected fromanhydrous and hydrophobic solvents, methylene chloride, or dimethylcarbonate, with the water soluble biologic being free of encapsulantsother than the organogel and the powder being dispersed in theorganogel, making a xerogel from the organogel, and providing thexerogel as a collection of particles, said collection being for deliveryto a patient, wherein the xerogel is a hydrogel upon exposure to anaqueous solution.
 39. The process of claim 38 wherein the organicsolvent is an anhydrous and hydrophobic solvent.
 40. The process ofclaim 38 wherein the organic solvent is methylene chloride.
 41. Theprocess of claim 38 wherein the organic solvent is dimethyl carbonate.42. The process of claim 38 wherein an amount of the water solublebiologic in the powder is at least 50% w/w.
 43. The process of claim 38wherein an amount of the water soluble biologic in the powder is atleast 80% w/w.
 44. The process of claim 38 wherein the powder consistsessentially of the water soluble biologic.
 45. The process of claim 38wherein the water soluble biologic comprises a member of the groupconsisting of an antibody, an antibody fragment, and a short chainvariable fragment antibody.
 46. The process of claim 38 wherein thewater soluble biologic comprises a protein or a fusion protein.
 47. Theprocess of claim 38 wherein the water soluble biologic comprises amember of the group consisting of a carbohydrate, a polysaccharide, anucleic acid chain, an antisense nucleic acid, a ribonucleic acid (RNA),a deoxyribonucleic acid, a small interfering RNA, and an aptamer. 48.The process of claim 38 wherein the water soluble biologic comprises ananti-vascular endothelial growth factor (VEGF) agent.
 49. The process ofclaim 38 wherein the water soluble biologic provides for treatment ofmacular degeneration.
 50. The process of claim 38 wherein the watersoluble biologic comprises an anti-cancer agent.
 51. The process ofclaim 38 wherein the water soluble biologic comprises insulin.
 52. Theprocess of claim 38 wherein the water soluble biologic comprises agrowth factor.
 53. The process of claim 38 wherein the water solublebiologic comprises a member of the group consisting of antibiotics,antineoplastic agents, hormones, angiogenic agents, anti-angiogenicagents, neurotransmitters, psychoactive drugs, chemotherapeutic drugs,drugs affecting reproductive organs, and genes.
 54. The process of claim38 wherein the water soluble biologic is for treatment of a conditionselected from the group consisting of elevated intraocular pressure,fungal blepharitis, conjunctivitis, keratitis, open-angle glaucoma,ocular hypertension, inflammation of an eye, keratoconjunctivitis, andallergic conjunctivitis.